eamag/protocols-with-steps huggingface dataset:
TITLE: Home-made TOP10 competent heat-shock cells
AUTHORS: Magdalena Julkowska

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The standard protocol to propagate TOP10 Competent cells for Heatshock transformation method</div></div>

[STEPS]
?. Grow a 5ml overnight culture of cells in LB media. In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/100..	Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)
?. Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at 4oC but if you have just made it fresh then put it in an ice bath).
?. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.
?. Centrifuge for 10 minutes at 3000 rpm and 4oC.
?. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.
?. Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
?. Add 100 μl aliquots to your chilled eppendorfs and store at − 80oC.The original paper [1] suggests freezing the cells immediately using a dry ice bath. I (BC) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at − 80oC also seems to work well (Jkm)If you run a control every time you clone (i.e. a vector-only ligation), you can as well freeze cells in 200 μl aliquots. Unused cells can be frozen back once and reused, albeit with some loss of competence.
?. It is a good idea to run a positive control on the cells.
TITLE: NGM Agar
AUTHORS: Adrien Assie, Buck Samuel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">NGM agar recipe from wormbook</div></div>

[STEPS]
?. Start with
[water]
?. NaCl
3 g
?. Peptone
2.5 g
?. Agar
17 g
?. Autoclave with stir bar
?. Cool to  (faster in water bath)
55 °C
?. Then add the following while stirring on heat plate (DO NOT OVERHEAT)
?. of  CaCl2 (sterile)
0.5 mL
?. of 5 mg/mL Cholesterol (dissolved in ethanol)
1 mL
?. of  MgSO4 (sterile)
1 mL
?. of  Potassium Phosphate Buffer, pH 6.0 (sterile)
25 mL
?. Pour into petri dishes using sterile technique60 mm dishes = About  per plate100 mm dishes = About  per plate
7 mL
20 mL
TITLE: EMb encystment medium preparation (500 mL)
AUTHORS: Carrie A Flynn, Barbara Kazmierczak

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Recipe for EMb encystment medium, which is used to induce synchronous encystment of </span><span style = "font-style:italic;">Acanthamoeba castellanii</span><span> trophozoites. Adapted from Neff protocol.</span></div></div>

[STEPS]
?. [Dry ingredients]
To 500 mL bottle, add: KCl (0.1 M) NaHCO3 (0.04 M) MgSO4 x 7H2O (8 mM) CaCl2 x 2H2O (0.4 mM) 2-Amino-2-methyl-1,3-propanediol (AMPD, 0.32 mM)-stir bar
3.728 g
1.68 g
0.986 g
0.03 g
0.017 g
We have found that AMPD is a superior amine buffer to Tris for this medium and maintains a constant pH over time. When Tris is used instead, the pH of the medium steadily decreases to 7 during storage, resulting in poor induction of encystment.
?. [Water]
Bring volume to  with dH2O and place on stir plate (unheated).
500 mL
?. [pH]
Measure pH using pH meter. Confirm that the pH is between 8 and 8.5--anything in this range works for encystment.
?. [Water]
Stir until dissolved.
?. [Sterilize]
Filter sterilize into sterile 500-mL plastic bottle using 0.22 µm filtration system.
Storage in plastic, not glass, is essential to maintaining the pH.
?. [Sterilize]
Store at .
0 Room temperature
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Protocol for labeling bacteria with N15. The bacterial DNA was made &ldquo;heavy&rdquo; so that community viral and host bacterial DNA could be distinguished from each other after sorting by using density gradient centrifugation.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Gravlax
AUTHORS: Monica Hassan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Gravlax is fresh salmon that’s been cured with a combination of salt, sugar, herbs and grated beetroot. </div></div>

[STEPS]
?. Peel  and grate it
[beetroots]
?. Chop
[dill]
?. Put  in a plastic food container and fully cover it with the mix from Step 5
[fresh salmon]
Salmon skin side should be down
?. Mix   with   and all ingredients you've prepared above in a bowl
[sea salt]
[sugar]
?. Ground
[black pepper]
?. Grate
[lemon zest]
?. Close the container and refrigerate it
?. Remove the mix and refrigerate   again
?. Serve it on a slice of sourdough bread. Enjoy!
TITLE: FCMPASS - Importing fcs files 
AUTHORS: Joshua Welsh, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the steps required to import fcs files for calibration using the FCMPASS software. This is one of a number of protocols in the pipeline for performing small particle calibration using the fcmpass software package.</div></div>

[STEPS]
?. Once a dataset has been created click the ‘Begin Calibration’ button.
?. Import fcs files by selecting the ‘+’ icon next to the ‘Files to calibrate’ table.
?. In the new window navigate to the folder containing the fcs files you wish to calibrate and select ‘OK’.
?. The fcs files and related metadata will now be imported.
?. If the folder contains fcs files that you do not wish to be calibrated, select them and click the ‘–‘icon. The metadata related to the remaining files will then be reprocessed.
?. The parameters e.g. SSC-H, SSC-A that are available in further steps of the software are those that are common to all the loaded fcs files. If files that are loaded do not have any common parameter names a selection will not be available in these steps.
?. Under the ‘Sample Type’ column all loaded files by default are listed as ‘Sample’. For the relevant files these can be adjusted to ‘SSC Calibration’ or ‘FL Calibration’ depending on what the sample was used for.
?. Once completed select ‘Next’.
TITLE: Cassiopea In Situ Hybridization
AUTHORS: Bailey Steinworth

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a protocol for </span><span style = "font-style:italic;">in situ </span><span>hybridization using Digoxigenin-labeled RNA probes to genes of interest. A separate protocol describes the design and synthesis of those probes. The purpose of this protocol is to visualize the spatial expression of genes.</span></div><div class = "text-block">This protocol was written up by Bailey M. Steinworth and is based on the following publications:</div><div class = "text-block">Wolenski FS, Layden MJ, Martindale MQ, Gilmore TD, Finnerty JR. 2013. Characterizing the spatiotemporal expression of RNAs and proteins in the starlet sea anemone, Nematostella vectensis. Nat. Protoc. 8:900–915.</div><div class = "text-block">Sinigaglia C, Thiel D, Hejnol A, Houliston E, Leclère L. 2018. A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species. Dev. Biol. 434:15–23.</div></div>

[STEPS]
TITLE: Genomic DNA extraction from diatom P. multistriata
AUTHORS: FRANCESCO MANFELLOTTO, monia teresa russo, Rossella Annunziata, Mariella Ferrante

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Genomic DNA extraction from diatom P. multistriata </div></div>

[STEPS]
?. Grow cells in 250 ml
?. filter coltures with 1.2 μm nitrocellulose membranes
?. rinse filter with 1 ml f/2 medium in a falcon and then move cells to a 2 ml eppendorf
?. Centrifuge at 6000 rpm for 5 minutes at 4 °C and remove medium
?. Add: 400 mg of 0.2-0.3 mm zirconia/silica diameter beads, 500 μl phenol.
?. Mix with vortex 30 Hz 3 times for 85 seconds, each time put sample in ice for 60 seconds before vortex.
?. Resuspend cells with 500 μl of TE buffer (10 mM TrisHCl pH 7.6 and 1 mM EDTA pH 8.0)
?. · Centrifuge at 10000 rpm for 5 minutes at 4°C.
?. Recover aqueous phase in new 1.5 ml eppendorf (about 600 μl)
?. Add 500 μl of PCI (Phenol:Chloroform: isoamyl alcohol 25:24:1 v/v) and mix by inversion.
?. Centrifuge at 10000 rpm for 5 minutes at 4°C
?. Move the aqueous phase in a new eppendorf and add 5 µl of RNase-A 10 mg/ml
?. Incubate at 37 °C for 30 minutes.
?. Add 500 μl di PCI (Phenol:Chloroform: isoamyl alcohol 25:24:1 v/v) mix by inversion.
?. Centrifuge at 10000 rpm for 5 minutes at 4°C.
?. Move the aqueous phase in a new 2 ml eppendorf and add:50 μl of 3 M NaAc (pH ± 5) 1 ml of ethanol 96% (- 20 °C) 2 μl glycogen (- 20 °C)
?. incubate over night at -20°C.
?. Centrifuge the overnight samples at 13000 rpm for 30 minutes at 4°C
?. discard aqueous phase
?. Wash the pellet by adding 1 ml ethanol 70% and mix gently by inversion
?. Centrifuge at 13000 rpm for 10 minutes at 4°C
?. discard aqueous phase
?. Wash the pellet by adding 1 ml ethanol 70% and mix gently by inversion
?. discard aqueous phase
?. Remove aqueous phase and dry pellet at RT for at least 20 minutes
?. Centrifuge at 13000 rpm for 10 minutes at 4°C
?. add 50 μl of Preheated TE 1X (pH 8) or sterile MilliQ water to pellet of DNAPreheat the TE pH 8 or sterile MilliQ water at 55 °C
?. incubate at 55 °C for 20 minutes
?. quantify DNA concentration by nanodrop or Qubit
?. in order to check DNA integrity, run a small amount of DNA with 1% agarose gel
?. The DNA is ready and store it at -20°C
TITLE: Immunoprecipitation
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the process of immunoprecipitation.

[STEPS]
SECTION: Steps
1. Collect the HeLa cells by trypsinization and wash the cell pellet with PBS once before cells are lysed in lysis buffer (100 mM KCl, 2.5 mM MgCl2, 20 mM Tris-HCl pH 7.4, 0.5% NP-40).
 
 
 
SECTION: Steps
2. Lyse the samples for 20 min on ice before cell lysates are cleared by centrifugation at 20000 x g, 10 min, 4 °C. 
SECTION: Steps
3. Protein concentrations of the cleared protein lysates are then determined with the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher) and equal amounts are incubated with beads. 
SECTION: Steps
4. Precoat the beads with GST (negative control), NIX-GST, or BNIP3-GST as described in the protocol for the microscopy-based bead assay. 
SECTION: Steps
5. Incubate the HeLa cell lysates 20 min with precoated beads. 
SECTION: Steps
6. In the morning, wash the samples five times in lysis buffer before the beads are either submitted for analysis by mass spectrometry or for analysis by SDS-PAGE and western blotting by resuspending the beads in protein loading dye, supplemented with 100 millimolar (mM) DTT, and boiled for 5 min at 95 °C.

Note that for mass spec samples, the NP-40 is omitted from the washing buffer.

SECTION: Steps
7. Load the samples on 4-12% SDS-PAGE gels (NP0322BOX, Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher). 
SECTION: Steps
8. Transfer the proteins onto nitrocellulose membranes (RPN132D, GE Healthcare) for 60 minat 4 °Cusing the Mini Trans-Blot Cell (Bio-Rad). 
SECTION: Steps
9. After the transfer, membranes are blocked with 5% milk powder dissolved in PBS-Tween (0.1% Tween 20) for 60 min at Room temperature. 
SECTION: Steps
10. Incubate the membranes 60 min at 4 °C with primary antibodies dissolved in the blocking buffer, wash three times for 5 min, and incubate with species-matched secondary horseradish peroxidase (HRP)-coupled antibodies diluted 1:10,000 in blocking buffer for 60 min at Room temperature.
SECTION: Steps
10.1. Wash for 5 min (1/3).
SECTION: Steps
10.2. Wash for 5 min (2/3).
SECTION: Steps
10.3. Wash for 5 min (3/3).
SECTION: Steps
11. Wash the membranes three times with PBS-T and processed further for western blot detection. 
SECTION: Steps
12. Incubate the membranes with SuperSignal West Femto Maximum Sensitivity Substrate (34096, Thermo Fisher) and imaged with a ChemiDoc MP Imaging system (Bio-Rad). 
SECTION: Steps
13. Analyze the images with ImageJ 47 (RRID:SCR_003070; https://imagej.net/).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/CviRI-Purification-From-XZ-6E-Virus-Infected-NC64A-er4bd8w" target="_blank">CviRI Purification From XZ-6E Virus Infected NC64A Chlorella</a>.

[STEPS]
?. 
?. 
?.
TITLE: Protocol for Nuclei/ Cell Isolation and 10X Genomics Fixed RNA profiling for Human Ovary
AUTHORS: Nicolas Martin

[DESCRIPTION]
This is the 10X Genomics protocol to fix, dissociate, and profile RNA from human ovary tissue. 

[GUIDELINES]
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).

[STEPS]
SECTION: Cell/Nuclei Isolation Protocol for Human Ovary
1. The protocol CG000553 REV B was used to fix, dissociate, and isolate cells/nuclei from frozen human ovaries with the following modifications: 

1) 0.2 mg / mL of Liberase TH was used for dissociation at Step 2b, Page 6.
2) Two extra "spin only" (i.e., steps 3 and 4 ) of the octodissociator protocol were run for each sample due to the presence of intact/ large tissue pieces at the end of the run at Step 2c, Page 6.   
3) Counts were performed using the Cellaca PLX Automated Cell counter (PN: PLX-SYS1) and ViaStain AOPI staining solution (PN: CS2-0106-5mL) at Step 2i on page 6. 

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/sample-prep/tissue-fixation-and-dissociation-for-chromium-single-cell-gene-expression-flex


SECTION: Chromium Fixed RNA Profiling Reagent Kits
2. The protocol CG000527 Rev E  was used to generate gene expression libraries from fixed cell/nuclei suspension inputs.

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/library-prep/chromium-single-cell-gene-expression-flex-reagent-kits-for-multiplexed-samples

Samples were multiplexed in batches of 16 using the user guide CG000565. 

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/library-prep/chromium-single-cell-gene-expression-flex-reagent-kits-for-multiplexed-samples
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>Resuspend the lyophilized QIAGEN Protease in 1.4 ml of Buffer FG3 (hydration buffer) and store at 2–8°C or in aliquots at –20°C</p>
<p> </p>
<p>Frozen blood should be thawed in a 37°C water bath</p>
<p> </p>
<p>For every 1 ml of blood, mix together 500 μl Buffer FG2 (denaturation buffer) and 5 μl reconstituted QIAGEN Protease</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: pMOA 189F-682R
AUTHORS: Roey Angel, Eva Petrova

[DESCRIPTION]
A PCR assay targeting the alpha subunit of the particulate methane monooxygenase gene (pmoA). 
This is a general assay for methane oxidising bacteria using primers 189F–682R.
Note: these primers might occasionally also amplify the amoA gene of ammonia-oxidising bacteria.
 
189F      GGN GAC TGG GAC TTC TGG   (target seq.  189 - 207) Holmes et al. (1995),
682R      GAA SGC NGA GAA GAA SGC (target seq. 664 - 682) Holmes et al. (1995).
 
Fragment size: 508bp.

[STEPS]
SECTION: PCR mixture
1.  
 
* Buffer contains 1.5 mM MgCl2 at final concentration
SECTION: PCR program
2. 1.  94◦C – 4′
2.  x 10 {
       a.  94◦C – 1′
       b.  62◦C – 1°C – 1’
       c.  68◦C – 1′
      }
 3.  x 25 {
        a.  94◦C – 1′
        b.  52◦C – 1′
        c.  68◦C – 1′
       }
 
4.  68◦C – 10'
TITLE: Lipid Annotation of MALDI IMS Datasets
AUTHORS: Elizabeth Neumann, Jamie Allen, Jeff Spraggins, Danielle Gutierrez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope: </div><div class = "text-block">Annotate lipid species detected by MALDI IMS analysis.</div><div class = "text-block">Expected Outcome:</div><div class = "text-block">A list of lipid identifications with low mass errors (<5 ppm) that correlate to ion images produced by MALDI IMS analysis.</div></div>

[STEPS]
?. Perform a quadratic recalibration of the IMS dataset using common well-characterized lipids:m/z  734.5694, 760.5851, 782.5670, 788.6164, 798.5410, 810.6007, 832.5827, and 848.5566Software used are DataAnalysis or Mmass, and ppm error calculated in excel.
?. Create mass list from the averaged, re-calibrated IMS data set.
?. Calculate ppm error associated with common lipids, such as [PC(32:0)+H]+ and [PC(34:1)+H]+, for mass errors.  If mass error is above 3 ppm, re-calibrate.
?. If LC-MS/MS has been performed, annotate each m/z value against the LC-MS/MS list, otherwise move to 4.1
?. Use LIPIDMAPS database to annotate each m/z value with the following search criteria:https://www.lipidmaps.org/resources/tools/bulk_structure_searches.php?database=COMP_DB1a. Positive Mode Adducts: [M+H]+, [M+H-H2O]+, [M+Na]+, [M+K]+1b. Negative Mode Adducts: [M+H]-, [M+Cl]-, [M+HCOO]-, [M+OAc]-2.   Specify Mass Tolerance: 0.005 m/z3.   Specify Chains: Even Chains Only4.   Sort: Delta
?. Calculate ppm error associated with each assignment and remove assignments with errors larger than 5 ppm, although most have errors lower than 3 ppm.
TITLE: Calculo área ImageJ
AUTHORS: Javiera Poblete

[STEPS]
?. Para el cálculo de área de las rocas se utilizó el programa ImageJ versión 1.8. Dentro del programa se abre la imagen deseada, se va a “seleccionar”, luego click en “Straight” y señala una medida de referencia (1 cm), luego seleccionar “Analyze” y posteriormente “Set scale”, donde se marcó “ know distance: 1;  Unit of legth: cm.” (Esto es para estandarizar las mediciones). Para ya calcular el área de cada roca se va a la opción de “Polygon section” y se bordea el área deseada, luego se hace click en “Analyze” y se visualizan los datos apretando “Measure”.
?. Para el cálculo del área ramoneada primero se recortó la foto, seleccionando toda la cara expuesta de la roca, luego de eso selecciona “Image” y luego “Crop”. Una vez recortada la roca se adjusta el tamaño mediante “Image” luego “Adjust” y escribiendo el ancho y largo deseado (730/2200) sin marcar la casilla de “constrain aspecto ratio”. Una vez hecho lo anterior se guarda como .jpg en la carpeta correspondiente. El segundo paso es el calculo del área del biofilm y la ramoneada, para ello se abre la foto a analizar y se dirige a “Image” luego “type” seleccionando 8 bits, hecho esto se dirige a “Image” después a “Adjust” y se hace click en “Threshold”, estando en esta opción se mueve los parámetros de B&W defiendo lo que es biofilm y lo que es ramoneado con los parámetros “dark background y stack histogram sin marcar. Cuando se obtiene el área seleccionada va hacia “Analyze particle” con los siguientes parámetros: 0 – inf; Circularity 0.00-1:00; Show Mask. Seleccionamos Display Results y luego Summarize donde se visualizará el área seleccionada.
TITLE: Clinical efficacy and safety of drug interventions for primary and secondary prevention of osteoporotic fractures in postmenopausal women: network meta-analysis followed by factor and cluster analysis
AUTHORS: Liangliang  Ding

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol for this network meta-analysis is detailed in the Attachment.</div></div>

[STEPS]
?. Clinical efficacy and safety of drug interventions for primary and secondary prevention of osteoporotic fractures in postmenopausal women: network meta-analysis followed by factor and cluster analysis
TITLE: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
AUTHORS: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur

[DESCRIPTION]
We adapted a previously-described method (Pantazis et al., 2022) for employing Piggybac transfection to stably express doxycycline-inducible NGN2 in human iPSCs. After stable integration of NGN2, proceed to differentiate iPSCs using protocol “iNeuron differentiation from human iPSCs.” 

[GUIDELINES]
Citations:

Pantazis, C.B., Yang, A., Lara, E., McDonough, J.A., Blauwendraat, C., Peng, L., Oguro, H., Zou, J., Sebesta, D., Pratt, G., et al. (2022). A reference induced pluripotent stem cell line for large-scale collaborative studies. BioRxiv 2021.12.15.472643.

[STEPS]
SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
1. Culture iPSCs in a 10 cm dish coated with Growth Factor Reduced Matrigel (Corning) and feed daily with Essential 8 media (ThermoFisher). 
SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
2. Passage iPSCs with warm Accutase into Essential 8 media with 10 micromolar (µM) ROCK inhibitor. Plate 800,000 iPSCs into one Matrigel-coated well of a 6-well plate.
SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
3. 3 - 6 hours after plating, cells should be healthy and attached. Perform transfection using Lipofectamine Stem and a 2:1 ratio of donor plasmid to transposase:

 

SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
4. Check for transfection efficiency (BFP-labeled cells) on the next day using fluorescence microscopy.
SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
5. 4320 min after transfection, select for transfected iPSCs with 0.5 1811 puromycin.

SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
6. Cryopreserve selected iPSCs with 

 

SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
4.1. Passage iPSCs with Accutase to a 10 cm dish when cells are confluent enough for splitting. 
 

SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
5.1. Confirm purity of surviving transfected cells with fluorescence microscopy. When population is pure, withdraw puromycin. 
SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
6.1. Proceed to culture and induction to neuronal fate using doxycycline (see “Protocol: iNeuron differentiation from human iPSCs”).
TITLE: Calcium chloride preparation of competent cells
AUTHORS: Moriah Beck

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Day 1]
Innoculate 1-2 mls LB (no antibiotics) with single colony of cells and incubate at 37 °C overnight with shaking.
?. [Day 2]
Use 1/100 volume of overnight to start culture. i.e. If you are growing up 50 mls of competent cells, than add 500 μL of starter.
?. [Day 2]
Grow cells to OD600 of 0.5-0.7 at 37 °C.
?. [Day 2]
Put flask of cells in ice water for 15+ minutes.
?. [Day 2]
Spin down cells at 5K rpm for 10 min in sterile 50 mL conical tube.
?. [Day 2]
Pour off supernatant and resuspend cells in 50 ml of 0.1M CaCl2 and keep on Ice for 1hr.
?. [Day 2]
Spin down cells at 5K rpm for 10 min.
?. [Day 2]
Pour off supernatant and resuspend cells in 5 ml of 0.1M CaCl2 ,15% glycerol
?. [Storage]
Snap freeze 100 μL aliquots in liquid nitrogen and store at -80 ºC.
?. [Autoclave Prior to Starting]
0.1M CaCl2 and 0.1M CaCl2 in 15% glycerol should be autoclaved before useLB mediatube and flask for LB growth