recursal/Fanatic-Fandom huggingface dataset:
Maaf

noun
1 1 pembebasan seseorang dr hukuman (tuntutan, denda, dsb) krn suatu kesalahan; ampun: minta --; 2 ungkapan permintaan ampun atau penyesalan: -- , saya datang terlambat; 3 ungkapan permintaan izin untuk melakukan sesuatu: -- , bolehkah saya bertanya;
ber·ma·af-ma·af·an v ampun-mengampuni; saling memberi ampun: pd hari Lebaran mereka -;
me·ma·afi v memberi ampun kpd; mengampuni: sudilah Tuanku - hamba yg hina ini;
me·ma·af·kan v memberi ampun atas kesalahan dsb; tidak menganggap salah dsb lagi: ia telah - kesalahanku;
ma·af-me·ma·af·kan v saling memberi maaf;
per·ma·af·an n perihal bermaaf-maafan;
pe·ma·af n orang yg rela memberi maaf (memaafkan);
pe·ma·af·an n proses, cara, perbuatan memaafkan; pengampunan
Johnny Thunder

Johnny Thunder is an Australian adventurer who travels with Dr. Kilroy and Pippin Reed to discover many treasures across the world.

## Website Description

Johnny is a full time adventurer and explorer from the Land Down Under (Australia!). He is brave, instinctive, determined and very creative. He always finds a way to solve problems and help people in need.

## The LEGO Movie Essential Guide Description

**Building style:** An expert in building for survival in the wilderness

**Additional file notes:** No hiding place is too dangerous or remote for this adventurer

## Section heading

Write the second section of your page here.
Necromancer

## Card Info

**{Skill}** If there are empty slots in your trays, place up to {Natural Energy} new figures corresponding to this unit's card in the empty slots following the rules for placing units with **Regenerate**.

Figure Stats - 2 Defense, 1 Wound Threshold

Figure Upgrade - Necromancer

Upgrade Type - Champion

Unit Type - Infantry

Faction - Waiqar the Undying

Cost - 3

## Available From

* Waiqar Infantry Command Unit Upgrade Expansion
K560 Media for BL21 Protein Expression

In 1.5L of MiliQ water:

30g Tryptone

22.5g Yeast Extract

12g NaCl

3g Na2HPO4 (Sodium Phosphate)

1.5g KH2PO4 (Potassium Phosphate)

Autoclave on liquid cycle.

Once cool to the touch, you can add antibiotic. Then add:

12.5mL of 20% glucose (filtered)

You can make 20% glucose by dissolving 8g of glucose in 40mL of water and then filtering it.
Edo tensei

Jutsu ini merupakan jutsu yang dipakai untuk menghadapi perang dunia ninja yang keempat. Pemiliknya adalah Kabuto, dia mengklaim bahwa Edo Tensei miliknya jauh lebih sempurna dibandingkan dengan Edo Tensei versi Orochimaru.

Jutsu ini akan memanggil jiwa orang yang sudah meninggal di dunia lain dengan menggunakan orang hidup sebagai tumbalnya. DNA dari jiwa orang tersebut juga sangat diperlukan. Jika semua syarat sudah dipenuhi maka pemanggilan jiwa orang yang sudah mati tersebut dapat dilakukan.

Ritual Edo Tensei
Ritual Edo Tensei
Edo Tensei versi Kabuto memang sangat luar biasa, Edo Tensei ini bisa beregenerasi jika tubuhnya diserang. Dan tidak bisa dihentikan walaupun pemilik jutsu ini meninggal. Ada beberapa cara yang dapat dilakukan untuk menghentikan jutsu ini yaitu menyegel mayat edo tensei atau menggunakan genjutsu kepada pemilik Edo Tensei untuk menghentikan jutsu ini.

Kelemahan dari Edo Tensei adalah :
- Jika Terkena Hantaman RasenPlanet Naruto, Mayat yang di edo tensei akan beregenerasi lebih lama (Chapter 558, Bab 1)
- Mayat yang di edo tensei dapat melepaskan diri dari kendali, jika terkena teknik koto amatsukami. contohnya: Itachi Uchiha
- Tidak dapat menghidupkan Mayat yang disegel. Ex : Shiki fujin
- Dikatakan bahwa edo tensei versi kabuto tidak ada kelemahan. Tapi pada Manga Naruto Chapter 591 madara ET mengatakan : Satu resiko untuk jutsu ini, yaitu jika makhluk yang dibangkitkan mengetahui segel jutsunya, dia bisa merusak perjanjian edo tenseinya. Sehingga para ET akan memiliki tubuh abadi, chakra yang tak terbatas dan tubuh yang bisa bergerak tanpa dikendalikan.
Minum

Minum Dulu Bro, Kita bersulang...^^...Adhynata~^•^√ (talk)>mi·num v 1 memasukkan air (atau benda cair) ke dalam mulut dan meneguknya: hampir setiap pagi ayahku -- susu; 2 mengisap: dilarang -- rokok di tempat penjualan bensin; -- madat;-- darah orang, pb banyak merugikan orang dengan mengambil keuntungan yang berlebih-lebih darinya; -- serasa duri, makan serasa lilin, tidur tak lena, mandi tak basah, pb hal orang yang sangat bersusah hati sehingga segala sesuatunya tidak berasa enak;

me·mi·num v minum;

me·mi·numi v memberi minum: kusir itu sedang - kudanya;

me·mi·num·kan v 1 memberi minum sesuatu kepada: ia - obat itu kepada anaknya; 2 menyuruh (meminta dan sebagainya) minum: ia hendak - kerbaunya ke danau;

ter·mi·num v 1 sudah diminum: dua botol bir - habis; 2 tidak sengaja diminum: minumanku - olehmu; 3 dapat diminum: jamu itu tidak - olehku karena pahitnya;

mi·num·an n barang yang diminum: di depot itu dijual -, rokok, dan obat-obatan;- campur minuman yang disiapkan menurut resep tertentu dengan mengaduk, mengacak, atau menggoncangkan dua (atau lebih) macam minuman (bahan); - keras minuman yang memabukkan, seperti bir, anggur, arak, tuak; - ringan minuman yang tidak mengandung alkohol, misalnya teh botol, teh kotak, gula asam;

pe·mi·num n orang yang suka (gemar) minum minuman keras
Jadi malu

adjective
1 1 merasa sangat tidak enak hati (hina, rendah, dsb) krn berbuat sesuatu yg kurang baik (kurang benar, berbeda dng kebiasaan, mempunyai cacat atau kekurangan, dsb): ia – krn kedapatan sedang mencuri uang; aku -- menemui tamu krn belum mandi; 2 segan melakukan sesuatu krn ada rasa hormat, agak takut, dsb: murid yg merasa bersalah itu -- menemui gurunya; tidak usah -- untuk menanyakan masalah itu kpd ulama; 3 kurang senang (rendah, hina, dsb): ia berasa -- berada di tengah-tengah orang penting itu;
-- bertanya sesat di jalan ( -- berdayung perahu hanyut; -- makan perut lapar), pb kalau tidak mau berikhtiar, tidak akan mendapat kemajuan; -- kalau anak harimau menjadi anak kucing (kambing), pb tidak sepatutnya kalau anak orang baik-baik atau pandai menjadi jahat atau bodoh; -- tercoreng pd kening, pb malu yg tidak dapat dihilangkan lagi krn sudah diketahui orang banyak; tidak tahu -- , ki tidak bermalu; tidak pernah merasa malu;
-- besar malu yg amat sangat; -- mata segan; hormat;
ma·lu-ma·lu a 1 tampaknya sangat malu; merasa malu: jangan -, pilihlah makanan yg kausukai dan makanlah sampai kenyang; 2 agak malu: ia masih - menemui tunangannya;
- bahasa ki agak malu sedikit; - kucing berpura-pura malu;
ber·ma·lu v mempunyai rasa malu: perbuatan yg tidak -;
me·ma·lui v 1 merasa malu thd (kpd): mereka tidak lagi - jagoan yg sudah tua itu; 2 menaruh hormat pd; menyegani: ia sangat - guru-guru yg pernah mengajarnya;
me·ma·lu·kan v 1 menjadikan (menyebabkan, memberi) malu: kelakuan anak itu sangat - keluarganya; 2 menganggap malu; malu tt sesuatu: ia - tindakannya;
mem·per·ma·lu·kan v membuat jadi malu;
pe·ma·lu n (orang) yg mudah merasa (yg mempunyai sifat) malu: gadis - itu selalu menunduk jika diajak berbicara;
ke·ma·lu·an 1 v mendapat malu: terpaksa kita kabulkan kehendaknya, supaya kita jangan -; 2 n hal malu; sesuatu yg menyebabkan malu; 3 n alat kelamin (laki-laki atau perempuan);
ke·ma·lu-ma·lu·an a agak malu; tersipu-sipu: ia mengulurkan tangannya dng -]]
Mendekatlah

video File:Placeholder aku ingin Selalu bersamamu....selamanya 19:10, March 15, 2017 (UTC)19:10, March 15, 2017 (UTC)19:10, March 15, 2017 (UTC)19:10, March 15, 2017 (UTC)19:10, March 15, 2017 (UTC)19:10, March 15, 2017 (UTC)19:10, March 15, 2017 (UTC)~~>

Write the first paragraph of your page here.

## Section heading

Write the first section of your page here.

## Section heading

Write the second section of your page here.
Noah

Dan terjadi lagi

Kisah lama yang terulang kembali
Kau temukan lagi
Dari cinta rumit yang kau jalani

Aku ingin kau merasa
Kamu mengerti aku mengerti kamu
Aku ingin kau sadari
Cintamu bukanlah dia

Dengar laraku
Suara hati memanggil namamu
Karena separuh aku
Dirimu

Ku ada di sini
Pahamilah kau tak pernah sendiri
Karena aku selalu

Di dekatmu saat engkau terjatuh

Aku ingin kau merasa
Kamu mengerti aku mengerti kamu
Aku ingin kau sadari
Cintamu bukanlah dia

Dengar laraku
Suara hati memanggil namamu
Karena separuh aku
Dirimu

Dengar laraku
Suara hati ini memanggil namamu
Karena separuh aku
Menyentuh laramu
Semua lukamu telah menjadi lirihku
Karena separuh aku
Dirimu
Protein Purification (Syringe/Pump)

## Prepare the column:

Column should always be left in excess buffer. Do NOT let beads dry out!!
Column will not dry out by gravity flow alone but because of surface tension of
the buffer. But need to be careful when using pump to force liquid over column
to never let the liquid run out and pump the column dry.

Uncap column, hook up to
either pump or syringe as desired (will take a long time via gravity flow
alone). Let column mostly clear of whatever buffer it was in.

Equilibrate column: wash
column with at least one column volume of your properly pH’ed buffer to
equilibrate.

## Run your sample:

Once column is
equilibrated, fill column with your sample. Place free end of tube in the rest
of your sample (if any) and place beaker w/your sample under the column to form
a continuous loop. Set pump to ~ 15. Be sure to watch for a few minutes to make
sure it is stable. If you pump too fast, the pressure may blow the top lid off
the column and pump your sample all over the floor in your absence.

Allow to cycle for 20-25 minutes to get all of your sample (or as much as will bind) onto the column.
CHECK EVERY FIVE MINUTES TO ENSURE COLUMN DOES NOT RUN DRY! Tubes should be
coiled 1x or more in the beaker to avoid them falling out and pulling air into
the beads.

Once sample is loaded,
remove free end of tube from beaker and let drain til mostly through.

## Wash column:

Fill column with Wash
Buffer. Allow to flow for a few seconds. Place free end of tubing in Wash
Buffer beaker, allow pump to flow Wash Buffer over column until all Wash Buffer
has been used (save ~1 mL for spectroscopic analysis blank later). Stop column
by capping bottom and top once nearly all Wash Buffer has entered column (but
do not wait til beads run dry!)

## Elute sample:

Fill column with Elution
buffer. At this point, incubate the elution buffer for 10 min. Then allow to
elute via gravity or use syringe if you are impatient.

For information on regenerating the column: **Streptactin Column Regeneration**
Murat Şimşek

Kenan

**Murat Şimşek** es uno de los personajes secundarios en la serie ***Elif***. Fue uno de los antagonistas secundarios de la primera temporada. Se vuelve bueno.

Su forma de vida era similar al de su padre, machista, prepotente y abusivo. Sin embargo, cuando se fue Elif de la casa, Murat salía con chicas que hacía cambiar su vida de vago sin oficio, se enamoró a tal punto de querer mejorar su vida. Sus relaciones acabaron mal con dos muchachas, la última relación positiva fue con Feride Çetin hasta cierto punto cuando ella desapareció. A pesar de todo siguió como persona honrada al igual que su padre.
Amset-Ra

Amset-Ra is an Egyptian pharaoh who ordered the creation of six treasures to help him conquer the world, but was defeated by rebels and sealed away.

## Website Description

Thousands of years ago, the wicked Pharaoh Amset-Ra ruled ancient Egypt and hoarded all the riches of the land inside his trap-filled Scorpion Pyramid…but he wanted much, much more. He was served by legions of immortal mummy warriors and stone guardians of unstoppable might, but even that was not enough. At his command, the greatest sorcerers in all of Egypt crafted six mystical treasures that, when gathered together, would grant the Pharaoh enough power to conquer the entire world. But before that could happen, a band of rebels stole away five of the treasures, and Amset-Ra and his pyramid were buried beneath the desert’s shifting sands. Now the Pharaoh is about to return, and only a team of brave adventurers has a chance of stopping him!

## The Legend of Amset-Ra

Thousands of years ago, Egypt was ruled by the cruel and mighty Pharaoh Amset-Ra. He had the power to control fierce beasts of the desert, and enchanted warriors sworn to serve him for all time.

But it was not enough. He commanded his sorcerers to create six magical treasures that would give him even more power; enough to conquer the whole world.

But before the Pharaoh could use the six treasures, they were stolen away. He sent his warriors to find them, but while they hunted the treasures, Amset-Ra was defeated and locked away inside his own trap-filled pyramid.

Now Amset-Ra sleeps, but one day he will awaken and claim his power. You who discover this table-it must be your quest to find the treasures and keep them safe. But beware, for the Pharaoh's servants still stand guard, waiting for their master to rise again.

## Section heading

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Mummy Warrior

Write the first paragraph of your page here.

## Website Description

The Pharaoh’s mummy warriors are driven by one all-consuming purpose: to find the lost treasures of Amset-Ra and return them to their master before he reawakens. They will do whatever it takes to fulfill their mission, and woe unto anyone or anything that gets in their way! The mummies may be strong, but their slow-witted single-mindedness makes them easy for a clever adventurer to trick or trap. Unfortunately, they tend to travel in packs!

## Section heading

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DominodalekLEGO Wiki

## Welcome to the Wiki

Welcome to the wiki. We’re a collaborative community website about your topic that anyone, including you, can edit. Click the edit button at the top of any page to get started!

## Describe your topic

Write a description about your topic. Let your readers know what your topic is about and add some general information about it.

## Latest activity

Photos and videos are a great way to add visuals to your wiki. Find videos about your topic by exploring Fandom's Video Library.

File:Placeholder
Lord Vampyre

Write the first paragraph of your page here.

## BBCW Website Description

Lord Vampyre is the fang-toothed ruler of the monster realm. A nocturnal creature, he sleeps during the day, hanging upside down or snuggled up in a coffin. His Bride is a deadly damsel and a master of hexes and poisons. The Lord also has several strange bat creatures, horrible winged henchmen who hang out in the castle’s nooks and crannies waiting to strike. He’s always up to no good. But his latest plan to eclipse the sun and inflict eternal darkness upon the world is perhaps his most vile yet…

**Lord Vampyre Facts:**

**Species:** Vampiro Brickbitious

**Origin:** He rules the Monster Realm from the Vampyre Castle

**Weaknesses:** Sunlight and garlic bread

**Powers:** Shape shifting into a bat and sleeping upside down

**Hobbies:** Organ music, dental hygiene, casketball

## Section heading

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Dr. Inferno

Dr. Inferno is the head of a villainous organisation, and a frequent adversary of the Agents. He is very rich, though it is unknown where the wealth comes from.

## Allies

As well as his Inferno Henchmen and Magma Drones, Dr. Inferno commands several allied villains. Dr. D. Zaster was an old friend of his.

## Section heading

Write the second section of your page here.
Golden Crystal

The Golden Crystal is one of Amset-Ra's six treasures, and provides the ability to control animals.

## Website Description

Guarded by the mighty Scarab, the Golden Crystal grants the Pharaoh the power to command all of the beasts of the desert, from the largest crocodile to the smallest beetle. Snakes, scorpions, vultures and jackals – all will obey his will when he summons them to crush his enemies!

## Section heading

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Colony PCR

Choose and mark 28 well-separated colonies on your plates (preferably from only the 50 uL plate but can use both if needed). Circle each colony and label with numbers 1-28.

Recipe - (mix all together in order given, aliquot into PCR tubes in 20 uL each)

Reagent Per 20 uL reaction For all 28 reactions:

For 9.5 uL water in each 20 uL reaction, add 266 uL (\*\*\*Add more, - 275-285 uL to account for pipetting error)

Primer 1 \*\*100 uM 0.25 uL 7 uL

Primer 2 \*\* 100 uM 0.25 uL 7 uL

2x Green MasterMix 10 uL 280 uL (found in the -30)

To each 20 uL aliquot:

Gently touch the tip of a pipet (pipet depressed to ~ 5 uL) to the requisite colony to get some cells on pipet tip. Carefully place pipet into PCR tube and pipet up and down 6-7 times to suspend cells in solution. Spin down.

PCR program:

 95
 Tm-5
 72
 Repeat Cycle 24-25x
 12

\*\* Don’t forget to record the volume of the reaction in the PCR program BEFORE running.

When PCR run is complete, run your samples using Gel Electrophoresis and look for bands of the estimated size. If a colony, or a few colonies, on your gel show the correct predicted length, this means theses colonies were successfully transformed.

The next steps for your colonies will be:

Growing Bacterial Colonies in LB

Sending out DNA for Sequencing
Gel Purification

Cut DNA out of gel using the UV-cutting tray in the printer room, make sure to use correct safety equipment.

Place in a 2 ml tube (you may need to use two or more tubes, so plan accordingly)

Add solubilization buffer to cover gel (protocol recommends that you use a specific volume of buffer per volume of gel)

Place on 55C heat block

Check and invert tube every 3-5 minutes until melted

Add all liquid to a column filter tube and centrifuge at max (discard liquid)

Wash 2X with 200ul wash
buffer (centrifuge 30s at max and discard liquid)

Dry spin centrifuge 1 min at max speed

Transfer column to new
1.5ml tube

Add 50ul elution buffer
(that was kept warm on 50C heat block)

Incubate at room X 10min

Centrifuge max speed X
3min

Check concentration and label tube with DNA construct (like APE file), date, and [concentration]
Glycerol Stock

Transform DNA to BL21
cells (-80 Box labeled 60, colorful tubes)

Place 50ul of cells in
1.5ml tube

Place 0.5ul of Correct
clone sequenced DNA in tube

Place on ice X 15min

Place in 42C bath X 50s
(Make sure there is diH20 in well used)

2min on ice

Add 500ul SOC (under
flame)

Place on shaker 37C
270-290RPM for 75min

Plate to pre-warmed (at
37C) plate with correct antibiotic

10ul on one plate and
25ul on other

Grow overnight at 37C

Pick one colony and grow
overnight on 37C shaker (5ml LB + 5ul antibiotic “1X” + colony DNA)

Place 900ul of solution
grown overnight + 900ul 50% glycerol stock in 1.8ml cryotube

Label tube

Place in -80 (in correct
box)
KOD PCR

Fill an ice bucket and get the KOD MM (hot start) from -30 freezer. Thaw the MM in 37℃ water bath and vortex. Collect all other necessary reagents (primers, DNA, etc.) and vortex before adding them together. Label PCR tubes.

Procedure for 50 uL reaction with two primers (Mainly used for plasmid DNA. For full length KOD protocol, click here):

Add the following:

* 25 uL KOD MM
* 1.5 uL 10 uM primer 1
* 1.5uL 10 uM primer 2
* Calculated amount (uL) needed for 10 ng of plasmid (Ex. If you have 2.4 ng/uL of DNA, you would need about 4.167 uL of DNA to achieve 10 ng of plasmid.)
* Calculated autoclaved H20 to reach 50 uL volume

KOD Program:

1. Polymerase Activation: 95℃ for 2 minutes
2. Denature: 95℃ for 20 seconds
3. Annealing: Lowest Primer Tm℃  for 10 seconds
4. Extension: <500 bp = 70℃  for 10 seconds/kb; 500-1000 bp = 70℃  for 15 seconds/kb; 1000-3000 bp = 70℃ for 20 seconds/kb; >3000 bp = 70℃ for 20 seconds/kb
5. Repeat steps 2-4: 20-40 cycles
Mini-Prep

**\*\*\*USE**
NEW ZYMO KIT. FOLLOW INSTRUCTIONS IN PAMPHLET, NOT “SHORTCUT” INSTRUCTIONS. THE
BELOW ARE INSTRUCTIONS FOR MINIPREP USING OLD QIAGEN REAGENTS AND COLUMNS.

1. Grow cells in 5 mL of LB with correct selection agent overnight at 37 deg C.
2. The next morning, spin down 2 mL of each culture at at a time in 2 mL tube for 1 minute at top speed in the large centrifuge. Aspirate out media, taking care not to disturb pellet. RESERVE 500 uL to 1 mL of culture for glycerol stocks.
3. Add 250 uL of Resuspension solution to cell pellet. Pipette VERY GENTLY up and down to resuspend cells. (\*\*\*It is crucial at this stage to not form bubbles, as this can cause loss of as much as 50% of your plasmid DNA).
4. Add 250 uL of Lysis buffer; invert several times to mix. Incubate at room temperature for 3 minutes.
5. Add 350 uL of Neutralization solution; invert exactly 5 times, no more, no less.(At this stage, a white cloudy substance should be visible; this is your genomic DNA/protein lysate).
6. Spin at max speed in the large centrifuge for 7 minutes. Transfer supernatant to a spin column, taking care not to incorporate any of the cloudy white protein.
7. Spin supernatant through the spin column (Can be done in the small tabletop spinner)
8. Wash TWICE with 750 uL of Wash buffer; spin thru w/ tabletop spinner each time.
9. After removal of second wash, dry column by spinning for 2 minutes at top speed in the large centrifuge.
10. Transfer spin column to a fresh 1.5 mL tube. Add 50 uL of Elution Buffer. Spin for 4 minutes in large centrifuge at top speed to elute plasmid DNA. Check concentration using the nanodrop.
Golden Nemes

The Golden Nemes is the most powerful of Amset-Ra's six treasures.

## Website Description

The last and most powerful of the Pharaoh’s treasures is hidden somewhere within his own immense Scorpion Pyramid. The Golden Nemes headdress grants Amset-Ra the power to see visions of the future, guiding him in his wicked quest to rule the world. Only with the Golden Nemes upon his head can the Pharaoh command the united power of the six lost treasures

## Section heading

Write the second section of your page here.
Protocols

ATPase Assay

ATPase Assay (written by: Danielle)

Bacterial Cell Transformation

Baculovirus Production with Bac-to-Bac (Invitrogen)

Cloning Steps

Colony PCR

DDB Pull Down

DPN1 Digest

Emulsiflex

Gel Electrophoresis

Gel Purification

Gibson Reaction

Growing Bacterial Colonies in LB

KOD PCR

Making Competent Cells (using Zymo Mix and Go)

Mini-Prep

PCR Purification

Pipetting Basics

Processing Clones from Whatman FTA Discs (From DGRC)

Protein Expression in Bacterial BL21 Cells

Protein Purification (Syringe/Pump)

Sequencing

Streptactin Column Regeneration

TIRF Microscopy

Transformation (Gibson)

Q5 PCR

Making de-tyrosinated Microtubule for TIRF by VASHI plus SVBP
Filter Compatibility

Always check if what you are filtering is compatible with the membrane/filter you are using. I found this out the hard way--nitrocellulose is not compatible with ethanol (it will shrink).

For further reading about filter compatibility and FAQs:

https://tools.thermofisher.com/content/sfs/brochures/Filtration-FAQs-June2013.pdf

https://www.corning.com/media/worldwide/cls/documents/CLS-FIL-004%20REV4%20DL.pdf
JalView Protein Alignment

You can download JalView software for protein alignment here.

### Once you download Jalview (and java), open the program.

If you have a windows computer, there may be issues opening it. You may have to go to your downloaded jalview program, left click on jalview program and select "open with". To find the program needed to open jalview, you will need to go to your computer, then programs-86 then find java and open using the program ''javaws". The program opens up sample windows, which you can exit out of.

### To create a new alignment:

To find protein sequences, I recommend using NCBIs protein search tool. You'll need to know the specific name of your protein (not just kinesin 1, but Kif 5B) for the search. You can filter by length of the protein and by species. From here you can download the FASTA sequence.

Select: File --> input alignment --> from textbox

You can then input the one letter sequences of each of your proteins in the text box and click new window when finished. \*Note: this does NOT align your sequences.

If you input a sequence from ApE, it will not have its FASTA name:

>OWK03308.1 KIF5B [Cervus elaphus hippelaphus]

You can easily create your own name from this information:

>Kin1-523 KIF5B [Homo Sapiens]

### To align:

After you have input your sequences and have opened a new window, you can then align the sequences:

Webservice --> alignment --> clustal --> with defaults

This will actually align the sequences and will show you the conserved regions among a family of proteins. The tallest yellow bars show the most conserved regions, while the shortest show the least.

### Analysis:

In addition to simply aligning the protein sequences, you can also add colors that tell you specific information about the amino acids in the sequences. For instance, you can select colour and then hydrophobicity to see essentially a heat map of polar and nonpolar residues. Some of the colors I'm not familiar with, but here is what I've noticed so far:

Buried Index: Blue is very buried and green is exposed.

Hydrophobicity: Red is hydrophobic and blue is hydrophilic

Percentage Identity: Shows heat map of identity, purple being identical

Clustal: I believe that clustal colors similarly behaving amino acids in the same color.

Additionally, to easily find regions that are NOT conserved you can select format --> show nonconserved and this will change the letters of the conserved regions into dashes.

### Secondary Structure:

In addition to aligning, you can also predict the secondary structure of your proteins.

Select all sequences and go to: web service --> secondary structure prediction --> Jnet secondary structure

### Determining Residue Numbers:

Say you find a region that you think is interesting when comparing Map7 and Ens and you want to determine the residue numbers. You must scroll over the starting and ending residues of Map7 and look in the bottom left corner for the residue number. Then you must do the same for Ens. Jalview does not show the residue number for both amino acid sequences above them.
Nanodrop

Protocol for using Nanodrop:

1. Select what you are measuring (ex: ssDNA, dsDNA)
2. Lift up the arm and add 1uL of your blank to the reader. **Remember to blank first using 1uL of the solution you used in your purification protocol. i.e. if you used water, use water as your blank. If you used buffer, use buffer.**
3. Clean the nanodrop reader with a kim-wipe
4. Place 1uL of your samples one at a time on the reader. Don't forget to clean in between!
Bacterial Cell Transformation

Materials Needed: 50 uL competent cells, DNA to transform (from Gibson or otherwise), ice, 42 deg water bath, 2 agarose plates w/ proper selection media, 500 uL SOC media, plate spreader, ethanol, bunsen burner.

1. Fill ice bucket, take to -80 deg freezer. Take out tube of competent cells \*\*\*For Gibson Transformations: Use the XL10 Gold cells in box labeled 50 (on bottom layer of left-most rack, second from top shelf). They are aliquoted in 50 uL aliquotes. \*For transformations requiring BL21 cells: cells are in box labeled 60 (on left-most rack, second from top shelf) See Protein Expression in BL21 Cells for the correct protocol.
2. If necessary, aliquot 50 uL of cells into fresh 1.5 mL tube. \*\*Mark in-use tube of cells with a dot on lid.
3. Add half of Gibson reaction mixture to competent cells.
4. Incubate on ice for 15-20 minutes. In the meantime: Refreeze rest of competent cells in -80. Fill up appropriate # wells in 42 deg heat block with water.
5. Heat shock cells in water-filled 42 deg heat block for 45 seconds.
6. Place back on ice for 2 minutes.
7. Add 500 uL SOC media \*\*Careful not to tip bottle or get any media in cap! And open next to a flame!
8. Shake tubes for 1 hours @ 37 deg C.
9. Grab the agarose plates from the refrigerator and place them in the incubator.

\*Make sure agarose plate contains the same resistant as the DNA being transformed.

1. If transforming a ligation or Gibson reaction where efficiency may be low, plate cells on 2 plates as follows: 50 uL onto one plate and rest of cells onto second plate. Spread w/spreader. If transforming a plasmid where efficiency should be high, plate 5-20uL. Place in incubator overnight.
2. Next morning, check plates. Remove plates from incubator when size of colonies is a little bigger than the tip of a pipet tip. Store in 4 deg fridge (parafilm for longer storage) if not using immediately.
Zeynep Şimşek

Zeynep

Zeynep Şimşek es uno de los personajes principales en la serie ***Elif***. Es la hija de Veysel y mejor amiga de Melek, con quien se crió. Es de buen corazón y está enamorada de Selim, su amor le parece imposible pero el tiempo le demuestra lo contrario.

Apareció hasta la temporada 3, y la última vez que se la vio fue como una vida cotidiana con su esposo Selim.

## Curiosidades

* En el doblaje latino fue doblada por Jessica Monzón, actual esposa de Nacho Pineda Díaz quien dobló a Selim, son esposos en la vida real.
ApE Software

You can download ApE, a plasmid editing software, here.

ApE is used to view and edit potential and current DNA sequences (such as plasmids, g-blocks etc.)

## Getting Started:

### How to view the sequence of a feature (primer, tag, etc.):

At the top of the page is a list of features of your current sequence. To highlight where each feature is in your DNA sequence, double click on the name of the feature you want to view in the list. Additionally, if you are moving your mouse over a certain sequence, the name of the features in that area will appear underneath the list of features at the top. This will help you to determine where one feature ends, and another begins, or where they overlap.

### How to add a new feature:

Highlight the sequence in the file, and right click and select add new feature. (Alternatively, you can highlight the sequence and go to the dropdown menu at the top of the page to add a feature.) After adding a feature, a new window will pop up. Change the color to something different than the surrounding colors, and make sure to name it something descriptive. (Note- DO NOT select reverse complement! This will change the actual sequence to the reverse complement and cause all sorts of problems!)

### How to determine Tm:

PCR usually requires that you know your Tm, or the melting temperature of the PCR product. For best results when designing primers, try to get the Tm's of primes in the same reaction within 5 degrees of one another, for best results. Usually, you'll pick the lowest Tm and subtract 5 degrees for best results with PCR amplification

With Gibson- Usually, with PCR for Gibson, you will design primers that extend farther than your DNA, to create overlaps between two sequences for Gibson. Therfore, when determining Tm for PCR, you will need to highlight the piece of the primer that will actually bind to the DNA, and exclude the overhang. At the top of ApE in the right corner will be "Tm= " which will tell you your correct Tm.

With general PCR- Simply highlight the entire primer and read the "Tm= " in the top right corner.
Making Competent Cells (using Zymo Mix and Go)

The following procedure
is for a 50 ml E. coli culture in ZymoBroth™ (supplied with

T3001 only) or SOB
medium; however, the volume can be adjusted according to your specific
requirements. I often do larger volumes in the Vale lab, using the larger kit
T3002.

Day 1: Autoclave centrifuge bottles, or if you will be using fresh sterile
bottles each time, please omit this step.

2.    Day
1 afternoon: Use a sterile 200 ul pipette tip to dip into a commercial
competent cell tube, and streak it onto a plain LB agar plate.  (Note this
step is not strictly required but maybe good to get a single genetically pure isolate
from the tube)

3.    Day
2 morning: Pick a single colony and inoculate 0.5 ml LB culture. Best to pick
and grow 2-3 colonies in separate tubes to give yourself multiple overnight
cultures at different dilutions.

4.    Day
2 evening: Inoculate the 0.5 fresh LB culture into 50mL ZymoBroth™ or SOB
medium in a 500 ml culture flask. Best to inoculate 2-3 different cultures at
different dilutions. Use 100, 250, and 500uL from starter tubes to do this.
This allows you to get multiple chances at getting to the correct OD the next
day in a reasonable time. Shake cultures vigorously (150 - 250 rpm) at 20 C
until OD600nm is 0.4 - 0.6. Note that E. coli grows very slowly at this
temperature. It usually take about 16 – 20 hours to reach the desired OD,
depending on the stain.

5.    Day
3, Buffer Preparation Prior to Harvesting the Cells…The Wash and Competent
Buffers are provided as 2X stock solutions. They need to be diluted to 1X by
adding an equal amount of Dilution Buffer. To prepare 5 ml of 1X Wash Buffer:
Add 2.5 ml Dilution Buffer and 2.5 ml of 2X Stock Wash Buffer. To prepare 5 ml
of 1X Competent Buffer: Add 2.5 ml Dilution Buffer and 2.5 ml of 2X Stock
Competent Buffer. Keep these freshly prepared 1X Buffers ice cold. It is
important that each step of the following procedure should be done on ice or at
0-4°C.

Pre-cool pipette tips in the cold room (Optional).

Transfer the culture from Step 3 to ice. After 10 minutes, pellet the cells by
centrifugation at 3,000 - 3,700 rpm (i.e., 1,600 - 2,500 x g) for 10 minutes at
0 – 4 C.

Remove the supernatant and resuspend the cells gently in 5 ml ice-cold 1X Wash
Buffer. Re-pellet the cells as in Step 2.

Completely remove the supernatant and gently resuspend the cells in 5 ml
ice-cold 1X Competent Buffer.

Aliquot cell suspension into sterile PCR tubes using a multi-channel pipette in
a cold room, or on ice.

Snap freeze the cells liquid N2 and store below -70 C for transformation at a
later time.
Clemen Larumbe

**Clemencia "Clemen" Larumbe Garrido** es una actriz de doblaje mexicana, que cuenta con una profesión de Psicóloga. Es hija de **Ismael Larumbe Sr.** y **Amparo Garrido** y hermana de **Ismael Larumbe** y **Armando Larumbe**. Nació en México, D.F. y actualmente reside en Cuernavaca, Morelos. Fue conocida por doblar al personaje de la Dra. Temperance 'Bones' Brennan en la serie de Fox **Bones**.
Streptactin Column Regeneration

**Streptactin is a mutated**
streptavidin protein so treat it like you would any other protein! **The most stringent way to wash is to use 0.5M NaOH. Fill the**
column and allow to drain by gravity. You must immediately neutralize the beads
with PBS or other neutral pH buffer after the NaOH has drained. Do 2 column
volumes to be sure beads are in the correct pH. Sometimes Rick includes 1%
Triton X-100 with the 0.5M NaOH to help remove any hydrophobic gunk.

Optionally, clean with
HABA buffer: 50mM Tris, pH8.0, 150mM K-ace, 1mM EGTA, 1mM HABA. HABA is orange
dye in solution but turns bright red when it binds to the streptactin. It binds
in the biotin/strep-tag binding pocket, so column will only turn red if the
streptactin protein is still competent to bind strep-tag. Wash with 1 column
volume of HABA buffer and then need to wash column with multiple columns of PBS
or other buffer to remove the HABA dye, until beads are mostly white again.
Sometimes they remain a little pink and that is ok.
Agar Plate Making

Need 20ml LB/Agar for
each plate

Read bottle for agar
concentration

Ie 40g/L

If you want to make 20
plates you’ll need a total volume of 400ml (make a little more for error) 425ml

40g/L X .425L = 17g LB +
425ml diH2O (to erlenmeyer flask)

Autoclave (liquid protocol)

Remove from autoclave
and cool to a temp where it is comfortable to touch flask (don’t want it to
solidify)

Add antibiotic ( need 1X
in total; start with 1000X stock)

If you have a total volume
of 425ml use M1V1=M2V2 to figure out how much antibiotic to add

(1000X)(Yml)=(1X)(425ml)

Y=.425ml remember to
convert to ul

.425ml X 1000ul/1ml=
425ul

Mix by swirling

Add approximately 20ml
to each plate

Mark plate with
appropriate color that corresponds to the particular antibiotic that was added

Colors:

Carbenicillin-Green

Kanamycin-Red

Chloramphenicol-Blue

Gentamycin-Purple

See antibiotic concentration page here
Olympus Confocal Microscope

Turn on
microscope/lasers/computer

    Prepare slide (see next
page for illustration)

a.
    Put four dots of vacuum
grease in a square

b.
    Put a dot of glycerol in
the center of the four dots

c.     Pick larvae into water to rinse off food

d.
    Dry larva off with Kimwipe

e.
    Put coverslip on and
squish slightly (larva should be vertical on the slide)

f.
     Move
the coverslip left and right to roll the larva so two veins are visible, and roll so that the vein on the right is just about to the middle

g.
    Move the coverslip up and down to stretch the larva

    Pick the objective (20X, 40X, 60X, etc.)

    Put some immersion oil
on the objective and place the slide upside down

    Bring the objective up until the immersion oil spreads slightly (about pencil eraser size)

    Press button to change to fine focus!

    Check settings on FV1000 control panel

a.
    Laser power should ideally be between 10-15% (for brighter samples, lower is fine ~5%)

b.
    HV should be set between 750-900 (600-900 range is okay)

c.
    Pinhole size: 110um

d.
    Pixels 1024x1024

e.
    Generally, want 0.5-1s/frame

f.
     Generally, image 5 min (600 frames for 0.5s/f or 300 frames for 1s/f)

g.
    For Z stack step size
should be 0.3-0.5um

    Turn on epifluorescence or BF (trans) and focus on larva

    Find the head and move
down to segment A2/3/4 (A3 the best) by focusing out and passing over three
sets of denticles (or, “dentricles” if you are Paige)

For live imaging: image A3 and A4 (one 5 min
movie per segment). For morphology, image A2/3/4.

a.
    Alternatively: if on 40X or 20X focus on neurons and differentiate segments by gaps in the arbor. The 3rd cluster of neurons is in the A1 segment

10.  When in the correct segment, focus on neuron and
arbor

11.  Move the field of view so that you can see the entire arbor.

12.  Press “XY repeat” and image will appear on
computer screen rather than through binoculars. Adjust if necessary

13.  To take a Z stack, focus out until the neurons are no longer visible and press “set”, the focus in (the opposite way) until the neurons are no longer visible and press the other “set” (one is start and one
is end of the Z stack)

14.  Pick stack size

15.  Stop the “XY Repeat” and choose “depth” under
“XY LZ1” button

16.  Let run and press complete when done.

17.  Save all images and put onto mcb server

Crude rendering of slide-making process by Paige

Close up of denticles. Larval body segments Will not look so dark

When all neurons are lit up with GFP, the lowest neuron in the cluster is Class 1.

In class 1-GFP specific larva (Gal4^221, UAS-MCD8-GFP), image the

lower neuron as it is the most morphologically consistent between segments and
larvae.
Pipetting Basics

* One’s place does not show on the scale

* DO NOT go below 200 microliters on the scale

* Third number on the scale is the ones place after a decimal point (numbers are red colored as

opposed to black)

#### 0.5 – 10 microliters

* Use **red** colored pipet tray
* Third number on the scale is the ones place after a decimal point
* USE between 2 – 10 microliters

#### 0.1 – 2 microliters

* Use **red** colored pipet tray
* 1s, .1s and .01s place is shown

\*\*NOTE: IF TIP OF PIPET TOUCHES CLOTHING, SKIN,
COUNTER, ETC. USE A NEW TIP AND TOSS OLD ONE OUT. \*\*

### **How to use a pipet:**

1. Unlock pipet by
turning blue knob from the lock label to the unlocked label.

2. Adjust the scale to
the amount you wish to pipet by turning the knob on the top.

3. Lock pipet by
turning blue knob back to locked label.

4. Push the bottom of
the pipet onto a tip until you are sure it is secured

5. Apply thumb on the
top knob of the pipet and push down until you full first resistance.

6. Apply the tip into
solution you wish to pipet but only enough to be able to obtain solution
securely.

7. Release the knob
while tip is in solution and then pull back the tip once you have released your
thump completely.

8. Hold the tube you
wish to pipet your solution into in your other hand in order for you to be able
to see what is happening up close.

9. Place the tip in the
tube and push down on the knob all the way in order to make sure all the
solution has exited the tip

10. Keep your hand pushed down on the knob
until you have released the tip from the solution to make sure none of the
solution gets sucked back up into the tip

11. Discard the tip in a non-hazardous waste tub.
Cloning Steps

10 Steps of Successful Cloning:

1. PCR vector and insert(s), verify with Gel, DPN1 digest, and then purify DNA
2. Gibson
3. Transform competent cells (two protocols listed: 1 and 2)
4. Plate cells on selective media
5. Plates have colonies
6. Colony PCR on 28 colonies, run gel
7. Pick 2 colonies, grow in LB
8. Miniprep
9. Sequence DNA
10. Smile!
Gel Electrophoresis

Refer to Agarose Gel making page for how to make the gel.

If you already have pre-made gel, follow the rest of the procedure:

1. Heat up your flask of agarose gel in the microwave and swirl occasionally to dissolve. Usually, about 1 minute of heating per 100mL of agarose is good place to start.  **Watch CAREFULLY** to make sure the liquid doesn’t boil over in the microwave. It is best to periodically take out and CAREFULLY swirl the flask to ensure all agarose dissolves (you can check by holding up to the light and looking for the disappearance of whitish/translucent chunks of agarose). When it is completely dissolved the liquid should be totally transparent.
2. Pour about 50mL (for thicker gels use 55-60mL) from the large flask into the smaller flask (200mL) at the gel pouring station.
3. Add 5uL EtBr\* and swirl the small flask before pouring into the gel cast. \***Make sure to dispose of tips that have contacted ANY EtBr in designated waste beaker at the gel station. (Note: EtBr is in the FPLC cold room, bottom drawer, in a white box).**
4. For colony PCR gels, set up the cast with two of the smaller combs. You will have exactly 30 wells for your 28 colony sample and an additional two for your ladder.
5. Pour gel into the cast and let solidify about 30 minutes. After solidification, if you only need half of your gel, please cut the bottom half for someone else to use! (optional: you may pour your gel in the 4C for faster solidification)
6. After solidification, remove the combs and place your gel in the gel box with TAE buffer in them. Make sure that TAE covers all of your wells.
7. Pipet your DNA ladder and samples in to wells, and be sure to note their location in your notebook.
8. You may set a timer on the Electrophoresis system by changing the voltage button from V --> Amps --> clock, and selecting the time, and then selecting the voltage that you want.
9. Run gel at 120V (the time will depend on the size of your expected product).
Cleaning A Clogged Column (NOT FPLC Column)

1. Transfer beads to new column.

a.  Check that new column
flows well by filling it with diH2O.  The column should be a continuous
stream when full and be a rapid drip at half full.

b. Transfer beads by resuspending in PBS and transfer PBS/beads slurry to new column. Resuspend/transfer in as little volume at a single time.

c.  Continue to resuspend
and transfer small amounts of PBS/bead slurry until all beads are transfered.

1. Wash column with diH2O until residual beads can no longer be seen.  Feel free to cap and shake the column to agitate beads stuck to the walls.
2. Attach the tip of the column to the diH2O spout.  The fit should be snug. Slowly turn on the diH2O tap until a steady stream comes out the top of the column.  Continue flushing in this direction for a minute or so.
3. Attach column to stand with a waste beaker underneath.  Apply 1X column volume of 10% SDS. Fill the column again and cap.  Incubate SDS on column for 5 minutes then let it flow out.
4. Column with water until no more bubbles can be seen.
5. Apply 2 column volumes of .5 M NaOH as in Step 4.  Then wash as in step 5.
6. Test column speed as in Step 1a.  Repeat steps 3-6 as many times as it needed.
Q5 PCR

Fill an ice bucket. Put all reagents on ice (take Q5 MM out of -30 freezer only when ready to use it,
and put back ASAP). Label PCR tubes.

To make a 50uL reaction
using two primers add, in this order:

* Enough uL sterile H20 to make 50 total uL.
* 2.5uL 10uM primer 1
* 2.5uL 10uM primer 2
* For every 100 uL reaction, add enough DNA to get 25 total ng DNA. (So, for 20 uL reaction, 5 ng DNA. Note: Q5 protocol suggests 1pg-1ng plasmid DNA in 50uL reaction.
* 25uL Q5 MM-- and pipet up and down to MIX WELL

Note: for gradient PCR
make one large MM and aliquot into PCR tubes for efficiency. (Note: both PCR machines can do gradients, but the ProFlex one can only do two temps, whereas the Bio-Rad can do eight.)

Vortex everything to mix
before using. Thaw the Q5 by gently warming in 37℃ water bath until not
quite completely thawed. Let the rest thaw at room temp (invert). Vortex to
mix. Centrifuge if necessary.

Q5 program:

 Activate Polymerase
 Anneal
 Extend
 Final Extension
 Hold

Run for 35-45X

Note: enter the lowest and highest times for gradient PCR and the
machine will automatically make intervals. Load samples in direction that
temperatures are shown on screen.

Only tighten the lid until slight resistance. When PCR program is
finished, press “cancel” and wait until fully cancelled to open the lid.
Measure concentration. Store at 4℃.
MOM Lab UC Davis Wiki

## Welcome to the MOM Labs UC Davis Wiki

Welcome to the M.O.M. Lab Wiki! If you are new to Wikia, please visit the Beginners Corner to learn how to edit and add new pages, photos and links. Also, everyone in the MOM Lab should feel free to add any missing information to benefit the next user.

This wiki contains: Protocols , Equipment, Reagents, and Software. If you are new to the MOM Lab, please consider visiting our Getting Started page. This wiki can be edited by anyone at anytime to add, improve or correct things. In order to upload photos, you'll need to create an account and verify your email address, but for other changes you do not need an account.

To add a link to a page, just click edit, highlight the text you want to link, then click the chain-link and type in the exact name of the wikia page you are trying to link. **Note** that this page will have to have exact spelling and capitalization in order to link correctly, and not make an entirely new page. To add outside links, highlight the section of text and then click the chain-link icon and post in the URL. If you are unsure how to edit links, please visit the Beginners Corner and try to avoid editing preexisting links.

If there is more than one way to perform a protocol, it is better to add a new page (option 2 or alternative protocol) rather than editing a preexisting page. This wiki is searchable, through the "Search This Wiki" search bar above. When making a page, consider using key words that will make it more searchable, ideally in the title. **Imagine that a new undergrad in the lab is reading your article or protocol. Will they need to come and ask you for more info or clarification? In a wiki you'd ideally like for the answer to this question to be "No"**

 If you want to add new Protocols, Equipment, Reagents, or Software, it is better to start by editing the index page for that topic in order to add the linked page. You'll have to start by typing the new page name on the index page (notice they are alphabetically ordered). Then you can highlight the page name and click the chain-link icon, the name of your new page will come up in Red. Once you save that new page, and then save the index page, you'll see your new page listed in red font. Once you click on the new page, you will be prompted to add text to the page you have created. Have any questions? Try visiting the Beginners Corner.

Also, feel free to visit the Ori-McKenney Lab page here.

## Latest activity

Photos and videos are a great way to add visuals to your wiki. Find videos about your topic by exploring Fandom's Video Library.
Transformation (Gibson)

**Transformation Protocol:**

1. Add half\* of your Gibson reaction to 50uL of cells. \***don’t exceed 5uL of Gibson reaction in 50ul of**

cells

1. Incubate on Ice for 20 min.
2. While incubating, fill the 42 heat-block with water
3. After the 20 minute incubation on ice, heat shock at 42degC for 50 sec
4. Place the tube directly back on ice
5. Add 500uL SOC medium and incu-shake at 37degC for 2 hrs.
6. Plate 50uL and incubate plates at 37degC incubator overnight.
7. In the morning, remove your plates and store in the 4C.