iGEM 2016 — Team Minnesota

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# Team:Minnesota/Notebook

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#

####

Please refer to our notebook document [here](https://docs.google.com/document/d/16xP046nxoG5p2qaM_Jbg69HKupGFCdeQDJ156RYgqr8/edit?usp=sharing)for a better formatted version of our notebook with gel images.

[>](#iqiuwy)**7/25**

##### PCR Instructions:

General PCR Amplification Formula:

- 1μL Forward Primer (10μM)

- 1μL Reverse Primer (10μM)

- 20ng DNA

- 1μL High Fidelity polymerase

- 25μL green mix (2x)

- Remaining volume up to 50μL of NF water

Recipe:

Mix together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last. Run PCR using the following generalized steps:

|  | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Temp. (°C) | 95 | 95 | 50 | 72 | Repeat Steps 2 - 4 | 72 | 10 |
| Time (min.) | 2:00 | 0:30 | 0:30 | 1:40 | 30 Times | 5:00 | Indefinitely |
|  |  | Denature | Anneal | Extend |  |  |  |

The DNA complex that is to be assembled began as two separate parts; each part was separately amplified through PCR and then stitched together through PCR.

Reaction 1:

- 1μL Construct 1a F (10μM)

- 1μL Construct 1a URA3 R (10μM)

- 2μL/20ng Construct 1a URA3 (10ng/μL)

- 1μL High Fidelity polymerase

- 25μL green mix (2x)

- 20μL of NF water

Reaction 2:

- 1μL Construct 1b R (10μM)

- 1μL Construct 1b URA3 F (10μM)

- 2μL/20ng Construct 1b URA3 (10ng/μL)

- 1μL High Fidelity polymerase

- 25μL green mix (2x)

- 20μL of NF water

##### Stitching Instructions

General Stitching PCR Formula:

- 1μL Forward Primer (10μM)

- 1μL Reverse Primer (10μM)

- 100ng DNA of each fragment

- 1μL High Fidelity polymerase

- 25μL green mix (2x)

- Remaining volume up to 50μL of NF water

Recipe:

Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last. Run PCR using the following steps, then open up PCR tubes, add primers, and continue PCR for 20 more cycles.

Stitching Reaction 1:

- 1μL Construct 1b R (10μM)

- 1μL Construct 1a F (10μM)

- 1μL/100ng Construct 1b URA3 (100ng/μL)

- 1μL/100ng Construct 1a URA3 (100ng/μL)

- 1μL High Fidelity polymerase

- 25μL green mix (2x)

- 20μL of NF water

The stitch reaction was put through PCR according to the above table. Agarose gel electrophoresis was later used on the PCR products to identify them. The resulting gel appeared as follows:

![](https://static.igem.org/mediawiki/2016/0/04/T--Minnesota--gel1.jpg)

Where the bands correspond to, from left to right: a 1Kb DNA ladder, the product of the stitch reaction, the product of reaction 1, and the product of reaction 2.

[>](#nxbfgr)**Notebook**

Research Notebook
Table of Contents:
7/25:PCR and Stitching protocol
7/26: Gel Extraction and SD URA plates (unsuccessful)
7/27: Preparing SD URA Plates
7/28: Yeast Transformation, PCR, Stitching
7/29: YPD Broth, Gel, Yeast and E.coli Transformation, PCR, Stitching
8/1: E.coli Transformations
8/2: Transform E.coli, SD URA Media, and all Stitching/PCR
8/3: Gel & Yeast Transformation
8/4: Gel, PCR
8/5: Gel, Mini
8/8: Digest psb1c3 and stitch
8/9: Streak, PCR
8/10: Stitch PCR, Streak
8/11: Gel and Liquid Culture
8/12: Miniprep, Digest, Gel
8/15: PCR Purification, Digest, Gel Extract
8/16:PCR Clean Up, URA3 Stitch, Gel
8/17: Gel, URA3 Stitch
8/18: Gel,Gel Extract, URA3 PCR
8/19: Gel, PCR
8/22: Gel, Gel Extraction, Stitch, PCR
8/23: Gel
8/24: Gel, Stitch, PCR
8/25: Gel, PCR
8/26: Gel, gBlock Amplification, PCR
8/29: Stitch, PCR Amplification
8/30: Gel
8/31: Gel
9/1: Stitch
9/7: Gibson Assembly
9/9
9/12
9/12: PCR, Gel
9/13: PCR
9/14: Gel, Gibson Assembly, PCR
9/15: Gel, PCR
9/16: Gel, PCR
9/19
Extended GA w/ 1+2 RD (w/ gel extract product)
Gel: gradient product        A:H          E: H had bands
9/23
Gel of RDI+2
9/27
Gel: Ladder, A, B, C, D (excised), E, F, G, H (excised)
9/28
PCR:
9/29
10/3
10/5
10/6
10/7
10/10
10/11
10/12
7/25:PCR and Stitching protocol
Calculating Dilution Volumes:
30nmol → 100μM = 100000nmol/L
30nmol  =  100000nmol               x = 0.0003L = 300μL   (or just multiply given nmol by 10)                                                                                                                                                        .   x                   1L
PCR Instructions:
Ingredients:
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
20ng DNA
1μL High Fidelity polymerase
25μL green mix (2x)
Remaining volume up to 50μL of NF water
Recipe
Mix together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last. Run PCR using the following steps:
1
2
3
4
5
6
7
Temp. (°C)
95
95
50
72
Steps 2-4
72
4 or 10
Time (min.)
2:00
0:30
0:30
3:00
(30-40)x
5:00
∞
Denature
Anneal
Extension
Reaction 1:
1μL Construct 1a F (10μM)
1μL Construct 1a URA3 R (10μM)
2μL/20ng Construct 1a URA3 (10ng/μL)
1μL High Fidelity polymerase
25μL green mix (2x)
20μL of NF water
Reaction 2:
1μL Construct 1b R (10μM)
1μL Construct 1b URA3 F (10μM)
2μL/20ng Construct 1b URA3 (10ng/μL)
1μL High Fidelity polymerase
25μL green mix (2x)
20μL of NF water
Stitching Instructions
Ingredients:
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
100ng DNA of each fragment
1μL High Fidelity polymerase
25μL green mix (2x)
Remaining volume up to 50μL of NF water
Recipe:
Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last. Run PCR using the following steps, then open up PCR tubes, add primers, and continue PCR for 20 more cycles.
1
2
3
4
5
6
7
Temp. (°C)
95
95
50
72
Steps 2-4
72
4 or 10
Time (min.)
2:00
0:30
0:30
1:40
(30-40)x
5:00
∞
Denature
Anneal
Extension
Reaction:
1μL Construct 1b R (10μM)
1μL Construct 1a F (10μM)
1μL/100ng Construct 1b URA3 (100ng/μL)
1μL/100ng Construct 1a URA3 (100ng/μL)
1μL High Fidelity polymerase
25μL green mix (2x)
20μL of NF water
Solving for how much water to add to dry DNA:
29.9nmol/XL=100uL
X=299uL
Gel: Ladder, Stitch, Gblocks
7/26: Gel Extraction and SD URA plates (unsuccessful)
G-block 1: mass=380mg; volume of buffer=1520μL\*
G-block 2: mass=240mg; volume of buffer=960μL
Stitch: mass=300mg; volume of buffer=1200μL
\*400μL per 100mg
Preparing SD URA Plates (incorrect recipe/procedure):
500mL\*
3.35g 0.67% nitrogen base w/o amino acids, w/o ammonium sulfate 1.7g
2.5g 5g/L ammonium sulfate
0.7g 1.4g/L Yeast Drop out supplement
10g 2% agar 20g
10g 20g/L glucose or Galactose for induction medium
8mL 10g/L histidine
6mL 10g/L leucine
5mL 5g/L tryptophan
481mL H2O
\*Something fell out of the solution; process repeated successfully on 7/27
50mL Working Concentration
500mg 10g/L histidine
500mg 10g/L leucine
250mg 5g/L tryptophan
3.299g 1M LiAc
LB \*Plates
125g LB powder
10g Agar
500mL MilliQ water
7/27: Preparing SD URA Plates
Preparing SD URA Plates: Ingredients/Steps
Fill 600mL beaker with 400mL MilliQ H2O
Add 0.85g Yeast Nitrogen Base w/o amino acids or ammonium sulfate (while stirring)
Add 2.5g 5g/L ammonium sulfate
Add 1.4g Yeast drop out supplement
Add 10g agar
Add 10g dextrose
Add MilliQ H2O up to 500mL in a graduated cylinder
Autoclave on liq30
Let cool in 55°C incubator
Add 8mL of 10g/L histidine stock
Add 6mL of 10g/L leucine stock
Add 5mL of 5g/L tryptophan stock
7/28: Yeast Transformation, PCR, Stitching
Yeast Transformation:
Skip, not necessary
Scrape 25μL of several fresh yeast from plate into 1μL of sterile H2O
Pellet cells for 5s at max. Speed
Resuspend cells in 1μL of 100μM LiAc and incubate at room temperature for 5 min. Meanwhile, boil your salmon sperm carrier DNA for 5 min.
Divide your 1μL resuspended cells evenly into one tube for each transformation (up to 5 reactions); remove the supernatant with pipet
Add sterile components in this order per reaction:
240μL 50% PEG 3350
36μL 1.0M LiAc
10μL 10mg/μL carrier DNA
64μL H2O
Add DNA:
10μL (or ½) of the digested vector
25μL insert
Possible controls are uncut vector (-ctrl and cut vector ctctrl)
Vortex for 1 min. to resuspend cells in transformation mix
Incubate at 42°C for 20 min.
Pellet cells for 10 sec. at top speed; remove supernatant with a pipet
Resuspend cells in 400μL of H2O
Rlate out 10μL of yeast cells on one plate and 200μL on another
Allow to incubate at 30°C for 2-3 days
Qubit
Add 198μL buffer
Add 1μL DNA
Add 1μL dye
Run machine, don’t change calibration
PCR
Ingredients:
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
20ng DNA
1μL High Fidelity polymerase
25μL green mix (2x)
Remaining volume up to 50μL of NF water
3 Runs=Triple Ingredients:
3μL Forward Primer (10μM)
3μL Reverse Primer (10μM)
1.5μg DNA at 41.6ng/μL
3μL High Fidelity polymerase
75μL green mix (2x)
Remaining volume up to 150μL of NF water
Recipe:
Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last. Run PCR using the following steps:
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
72
Steps 2-4
72
4 or 10
Time (min.)
2:00
0:30
0:30
3:00
(30)x
5:00
∞
Denature
Anneal
Extension
PCR
1μL of each primer
2μL DNA @ 10ng/μL
25μL GoTag
1μL High Fidelity polymerase
20μL NF water
Stitching:
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
100ng DNA of each fragment
1μL High Fidelity polymerase
25μL green mix (2x)
Remaining water up to 50μ
7/29: YPD Broth, Gel, Yeast and E.coli Transformation, PCR, Stitching
YPD Broth:
5g powder
100mL MilliQ water
Gel: Ladder, AdH1 Stitch, 1A, 1B
120V for 30 minutes
Yeast Transformation:
Protocol 7/28
Reaction 1: Stitch 1 URA (333μL)
Reaction 2: Stitch 2 UldH1 (333μL)
Reaction 3: gblocks AldH1 (333μL)
E.coli Transformation:
Rb competent E.coli (uQ950)
Add DNA (no more than 20μL)
Incubate 30 min. on ice
Incubate @ 42°C for 1 min.
Incubate 2 minutes on ice
Incubate for 1 hour @ 37°C shaker; 750μL RB
Plate 1:1, 1:10, 1:100
PCR
Ingredients:
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
20μL DNA @ 10ng/μL
1μL High Fidelity polymerase
25μL GoTag
20μL of NF water
1
2
3
4
5
6
7
Temp. (°C)
95
95
50
72
Steps 2-4
72
12
Time (min.)
2:00
0:30
0:30
2:00
30x
5:00
∞
Stitching:
Ingredients:
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
100ng DNA of each fragment
1μL High Fidelity polymerase
25μL green mix (2x)
Remaining volume up to 50μL of NF water
Recipe:
Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last. Run PCR for 10 cycles, then open up PCR tubes, add primers, and continue PCR for 20 more cycles.
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
72
Steps 2-4
72
12
Time (min.)
2:00
0:30
0:30
3:00
30x
5:00
∞
8/1: E.coli Transformations
E.coli Transformations:
Thaw DNA and cells
Add 4μL @ 50pg/μL psBlC3
Incubate in ice for 30 min.
Incubate in 42°C for 1 min.
Incubate in ice for 2 min.
In test tube add 750μL RB and cells with DNA
Incubate in 37°C for one hour
Plate at 1, 10-2, and 10-4
Replate Yeast:
Scrape Yeast
Spread on new plate at different dilutions
Approx. 1x and 1x10-2x
100μL/plate on SD URA-
Gel Electrophoresis Order:
Ladder, PCR1, Stitch, 1A, 1B
Stitching
Ingredients:
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
100ng DNA of each fragment (1A and 1B ADH1)
1μL High Fidelity polymerase
25μL green mix (2x)
Remaining volume up to 50μL of NF water
Recipe:
Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last. Run PCR for 10 cycles, then open up PCR tubes, add primers, and continue PCR for 20 more cycles.
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
72
Steps 2-4
72
12
Time (min.)
2:00
0:30
0:30
3:00
30x
5:00
∞
PCR
Ingredients:
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
20μL DNA (1B AdH1) @ 10ng/μL
1μL High Fidelity polymerase
25μL GoTag
20μL of NF water
1
2
3
4
5
6
7
Temp. (°C)
95
95
50
72
Steps 2-4
72
12
Time (min.)
2:00
0:30
0:30
2:00
30x
5:00
∞
8/2: Transform E.coli, SD URA Media, and all Stitching/PCR
Making Gel Stock:
4g agarose and 400mL TAE 1 x Buffer
Heat in microwave until dissolved
E.coli Transformations:
Thaw DNA and cells
Add 4μL @ 50pg/μL psBlC3
Incubate in ice for 30 min.
Incubate in 42°C for 1 min.
Incubate in ice for 2 min.
In test tube add 750μL RB and cells with DNA
Incubate in 37°C shaker for one hour
Plate at 1, 10-2, and 10-4
Gel Order: Ladder, 1B, Stitch
Preparing SD URA Plates: Ingredients/Steps
Fill 200mL beaker with 400mL MilliQ H2O
Add 0.34g Yeast Nitrogen Base w/o amino acids or ammonium sulfate (while stirring)
Add 1.0g ammonium sulfate
Add 0.56g Yeast drop out supplement
Add 4.0g dextrose
Add MilliQ H2O up to 192mL in a graduated cylinder
Seperate into two 96mL portions
Autoclave both on liq30
Let cool in 55°C incubator
Add 1.6mL of 10g/L histidine stock
Add 1.45mL of 10g/L leucine stock
Add 1mL of 5g/L tryptophan stock
Stitching (AdH1 and URA3)
Ingredients
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
100ng DNA of each fragment (1A and 1B ADH1)
1μL High Fidelity polymerase
25μL green mix (2x)
Remaining volume up to 50μL of NF water
Recipe:
Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last. Run PCR for 10 cycles, then open up PCR tubes, add primers, and continue PCR for 20 more cycles.
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
72
Steps 2-4
72
12
Time (min.)
2:00
0:30
0:30
3:30
30x
5:00
∞
PCR ( 1A and 1B AdH1; 1A and 1B URA3)
Ingredients:
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
20μL DNA @ 10ng/μL
1μL High Fidelity polymerase
25μL GoTag
20μL of NF water
1
2
3
4
5
6
7
Temp. (°C)
95
95
50
72
Steps 2-4
72
12
Time (min.)
2:00
0:30
0:30
2:00
30x
5:00
∞
8/3: Gel & Yeast Transformation
Gel Order: Ladder, 1A AdH1, 1B AdH1, AdH1 Stitch, 1A URA3, IB URA3, URA3 Stitch
Ran 120 V for 30 min
Yeast Transformation:
Skip, not necessary
Scrape 25μL of several fresh yeast from plate into 1μL of sterile H2O
Pellet cells for 5s at max. Speed
Resuspend cells in 1μL of 100μM LiAc and incubate at room temperature for 5 min. Meanwhile, boil your salmon sperm carrier DNA for 5 min.
Divide your 1μL resuspended cells evenly into one tube for each transformation (up to 5 reactions); remove the supernatant with pipet
Add sterile components in this order per reaction:
240μL 50% PEG 3350
36μL 1.0M LiAc
10μL 10mg/μL carrier DNA
64μL H2O
Add DNA:
Added 25μL of 1A URA3 and  25μL of 1B URA3 to one reaction
Added  25μL of stitch URA3 to other
Vortex for 1 min. to resuspend cells in transformation mix
Incubate at 42°C for 20 min.
Pellet cells for 10 sec. at top speed; remove supernatant with a pipet
Resuspend cells in 400μL of H2O
Rlate out 10μL of yeast cells on one plate and 200μL on another
Allow to incubate at 30°C for 2-3 days
8/4: Gel, PCR
Gel Order: Ladder, 1A AdH1\*, 1B AdH1, 1A ADH1, 1B ADH1\*, 1B ADH1
Ran 120 V for 30 min
E.coli Culture
5mL LB media
5μL chloramphenizol
Incubate at 37°C shaker overnight
PCR: 1B AdH1
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
2μL DNA (1B AdH1)
1μL High Fidelity polymerase
20μL GoTag
20μL of NF water
Recipe:Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last.
1
2
3
4
5
6
7
Temp. (°C)
95
95
54
72
Steps 2-4
72
12
Time (min.)
2:00
0:30
0:30
2:00
30x
5:00
∞
PCR: 1A AdH1
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
2μL DNA (1A AdH1)
1μL High Fidelity polymerase
20μL GoTag
20μL of NF water
Recipe:Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last.
1
2
3
4
5
6
7
Temp. (°C)
95
95
58
72
Steps 2-4
72
12
Time (min.)
2:00
0:30
0:30
2:00
30x
5:00
∞
1A and 1B ADH PCR:
8/5: Gel, Mini
Gel Order: Ladder, 1A AdH1, 1B AdH1; run at 120V for 30 minutes
Mini:
Add 1mL of E.coli to 1.5mL tubes
Pellet E.coli by centrifuging 2 minutes at 9000rpm; dump out excess media
Add 250μL of resuspension buffer (R3) to pellet and resuspend
Add 250μL Lysis Buffer (L7), invert tube 5 times, and let incubate at room temperature for 5 minutes
Add 305μL precipitation buffer (W4) and invert 5 times; centrifuge 10 minutes at 12000rpm
Load supernatent into a spin column and centrifuge @ 12000rpm for one minute; discard flow through
Add 500μL wash buffer (W10) incubate at room temperature for one minute, centrifuge at 12000rpm for one minute, discard flow through
Add 700μL wash buffer (W9) and centrifuge at 12000rpm for one minute, discard flow through
Centrifuge again for one minute at 12000rpm, discard flow through
Place spin column in 1.5mL tube, add 75μL of TE buffer (TE) and incubate column for one minute at room temperature
Centrifuge column 12000rpm for 2 minutes, discard column and you have your DNA in the 1.5mL tube
8/8: Digest psb1c3 and stitch
Psb1c3 Digest:
5.4μL DNA (at 187ug/mL; gives 1000ng DNA)
1μL EcoR1 HF
1μLSpe1
2μL cutsmart
10.6μL NF water (up to 20μL)
Stitch Digest:
14.2μL DNA (at 70.4ug/mL; gives 1000ng DNA)
1μL EcoR1 HF
1μLSpe1
2μL cutsmart
1.8μL NF water (up to 20μL)
For both digests, add largest to smallest volume, adding enzymes (Spe1 and EcoR1) last
Gel:
Ladder, stitch, psB1c3
8/9: Streak, PCR
Streak:
2808
2809
2996
2937
2988
PCR: 1A and 1B AdH1
Primer: 1A pAdH1, 35.8nmol + 358μL NF water = R
Primer: 1B pAdH1, 25.6nmol + 256μL NF water = F
PCR: 1B AdH1
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
2μL DNA (1B AdH1)
1μL High Fidelity polymerase
25μL GoTag
20μL of NF water
Recipe:Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last.
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
72
Steps 2-4
72
12
Time (min.)
2:00
0:30
0:30
2:00
30x
5:00
∞
PCR: 1A AdH1
1μL Forward Primer (10μM)
1μL Reverse Primer (10μM)
2μL DNA (1A AdH1)
1μL High Fidelity polymerase
25μL GoTag
20μL of NF water
Recipe:Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last.
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
72
Steps 2-4
72
12
Time (min.)
2:00
0:30
0:30
2:00
30x
5:00
∞
8/10: Stitch PCR, Streak
Stitch:
1.25μL 1A F/1B AdH1 F
1.25μL 1B R/1A AdH1 R
1μL High Fidelity polymerase
25μL GoTag
20.5μL of NF water
1
2
3
4
5
6
7
Temp. (°C)
95
95
56
68
30x
68
4
Time (min.)
2:00
0:30
0:30
2:00
30x
5:00
∞
Streak: 2987 and 2996
8/11: Gel and Liquid Culture
Gel: Ladder, 1B, 1B, 1A, 1A
Liquid Culture:
5mL
5μL cm
colony
8/12: Miniprep, Digest, Gel
Miniprep:
Elute with 25μL TE next time (see 8/5 for procedure)
Digest Rxn 1:
17μL DNA @ 62.5μg/μL
1μL Ecor1 HF
2μL cutsmart buffer
Digest Rxn 2:
17μL DNA @ 62.5μg/μL
1μL Spe1
2μL cutsmart buffer
Digest Rxn 3:
16μL DNA @ 62.5μg/μL
1μL Ecor1 HF
1μL Spe1
2μL cutsmart buffer
Repeat these digests with next sample of DNA with same volumes
Rxn 4: EcoR1
Rxn 5: Spe1
Rxn 5: ExoR1 and Spe1
Gel: Ladder, 1, 2, 3, 4, 5, 6, 2987, 2996
8/15: PCR Purification, Digest, Gel Extract
PCR Purification (all stitch):
Add 300μL of DNA binding buffer to each sample (5x volume, each sample 60μL)
Load into spin column in collection tube
Centrifuge > 10000xg for 30 seconds, discard flow through
Add 200μL DNA wash buffer to each centrifuge for 30 seconds, repeat this step and discard flow through each time
Place column into new 1.5mL tube
Add > 6μL of DNA elution buffer directly to column, then centrifuge; DNA is in flow through
Digest:
    Digest Rxn 1:
43μL DNA @ 96.5μg/μL
1μL Ecor1 HF
1μL Spe1
5μL cutsmart buffer
Digest Rxn 2 and 3:
3μL DNA @ 96.5μg/μL
1μL Spe1/EcoR1
2μL cutsmart buffer
14μL NF water
Incubate at 37°C for one hour
Stitch Gel Extract:
See protocol
800μL dissolving buffer
25μL NF water instead of elution buffer
(all at 6.87ng/μL)
8/16:PCR Clean Up, URA3 Stitch, Gel
PCR Clean Up of Stitch using BioBasic kit (140μL PCR product): see kit for instructions
Gel: Ladder, 10μL stitch product
URA3 Stitch (1,2,3)
Ingredients
1μL 1A F
1μL 1B R
1μL 1A URA3 @100ng/μL
1μL 1B URA3 @100ng/μL
1μL High Fidelity polymerase
25μL GoTag
20μL of NF water
Recipe:
Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last. Run PCR for 10 cycles, then open up PCR tubes, add primers, and continue PCR for 20 more cycles.
8/17: Gel, URA3 Stitch
URA3 Stitch (4)
Ingredients
1μL 1A F
1μL 1B R
1μL 1A URA3
1μL 1B URA3
1μL High Fidelity polymerase
25μL GoTag
20μL of NF water
URA3 Stitch (5)
Ingredients
1μL 1A F
1μL 1B R
3μL 1A URA3 gel extract
3μL 1B URA3 gel extract
1μL High Fidelity polymerase
25μL GoTag
16μL of NF water
URA3 Stitch (6)
Ingredients
1μL 1A F
1μL 1B R
3μL stitch @ 6.87
1μL High Fidelity polymerase
25μL GoTag
19μL of NF water
URA3 Stitch (7)
Ingredients
1μL 1A F
1μL 1B R
1μL DNA
1μL High Fidelity polymerase
25μL GoTag
21μL of NF water
URA3 Stitch (-)
Ingredients
1μL 1A F
1μL 1B R
25μL GoTag
23μL of NF water
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
72
30x
72
12
Time (min.)
2:00
0:30
0:30
2:00
30x
5:00
∞
Gel: Ladder, URA3 Stitch (3 samples)
8/18: Gel,Gel Extract, URA3 PCR
Gel Order: Ladder, 4, 5, 6, 7, -
Gel Order: Ladder, 5
PCR: URA3 (see 8/17 for PCR conditions)
1μL 1A F
1μL 1B R
4.3μL DNA (20ng @ 4.7ng/μL)
1μL HF polymerase
25μL green mix
17.7μL NF water
8/19: Gel, PCR
Gel Order: Ladder, 5 (see 8/17)
PCR:
Rxn 1:
1μL 1A F
1μL URA3 R
2μL 1A URA3 (DNA)
1μL HF polymerase
25μL green mix
20μL NF water
Rxn 2:
1μL 1B URA3 F
1μL 1B R
2μL 1B URA3 (DNA)
1μL HF polymerase
25μL green mix
20μL NF water
Rxn 3:
1μL 1A F
1μL URA3 R
2μL gblock 1 (DNA)
1μL HF polymerase
25μL green mix
20μL NF water
Rxn 4:
1μL 1B URA3 F
1μL 1B R
2μL gblock 2 (DNA)
1μL HF polymerase
25μL green mix
20μL NF water
1
2
3
4
5
6
7
Temp. (°C)
95
95
50
72
30x
72
12
Time (min.)
2:00
0:30
0:30
2:00
30x
5:00
∞
8/22: Gel, Gel Extraction, Stitch, PCR
Gel Order: Ladder, 1a, 2a, 3a, 4a, 1b, 2b, 3b, 4b
Gel Extraction (Invitrogen Kit):
Add Gel Solubilization Buffer (L3) to the excised gel: Gel Tube Buffer L3 Volume ≤2% agarose 1.7-mL polypropylene 3:1 (i.e., 1.2 mL Buffer L3: 400 mg gel piece)
Place the tube with the gel slice and Buffer L3 into a 55°C incubator for 10 minutes.
After the gel slice appears dissolved, incubate the tube for an additional  5 minutes.
For optimal DNA yields, add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.
Purify the DNA using a Centrifuge
Pipet the dissolved gel piece onto a Quick Gel Extraction Column inside a Wash Tube. Use 1 column per 400 mg of agarose gel.
Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
Add 500 µL Wash Buffer (W1) containing ethanol to the column.
Centrifuge the column at >12,000 × g for  1 minute. Discard the flow-through and place the column into the Wash Tube.
Centrifuge the column at maximum speed for 1–2 minutes. Discard the flow-through.
Place the column into a Recovery Tube. Add 50 µL Elution Buffer (E5) to the center of the column.  Incubate the tube for 1 minute at room temperature.
Centrifuge the tube at >12,000 × g for 1 minute.
The elution tube contains the purified DNA.  Store the purified DNA at 4°C for immediate use or at −20°C for long-term storage.
URA3 Stitch (see 8/17 for PCR conditions)
Rxn 1:
1μL 1A F
1μL 1B R
2μL 1A URA3 ( @ 46.1ng/μL)
7μL 1B URA3 ( @ 14.0ng/μL)
1μL HF polymerase
25μL green mix
13μL NF water
Rxn 2:
1μL 1A F
1μL 1B R
8μL 1A URA3 ( @  12.7ng/μL)
8μL 1B URA3 ( @ 12.1ng/μL)
1μL HF polymerase
25μL green mix
6μL NF water
Recipe: Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last. Run PCR for 10 cycles, then open up PCR tubes, add primers, and continue PCR for 20 more cycles.
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
72
30x
72
12
Time (min.)
2:00
0:30
0:30
3:10
30x
5:00
∞
PCR: gBlocks 1 and 2 (aliquot each rxn into 5 pcr tubes)
Rxn A:
5μL 1A F
5μL URA3 R
6μL 1A URA3
5μL HF polymerase
125μL green mix
105μL NF water
Rxn B:
5μL 1B URA3 F
5μL 1B R
6μL 1B URA3
5μL HF polymerase
125μL green mix
105μL NF water
1
2
3
4
5
6
7
Temp. (°C)
95
95
50
72
30x
72
12
Time (min.)
2:00
0:30
0:30
1:30
30x
5:00
∞
8/23: Gel
Gel Order: Ladder, A, B, 1, 1, 2, 2
URA3 Stitch (see 8/17 for PCR conditions)
Rxn 1:
1μL 1A F
1μL 1B R
8μL 1A URA3 ( @ 12.7ng/μL)
7μL 1B URA3 ( @ 14.0ng/μL)
1μL HF polymerase
25μL green mix
7μL NF water
Rxn 2:
1μL 1A F
1μL 1B R
2μL 1A URA3 ( @  46.1ng/μL)
8μL 1B URA3 ( @ 12.1ng/μL)
1μL HF polymerase
25μL green mix
12μL NF water
Recipe: Mix everything except primers together into PCR (small) tube starting with the largest volume and working your way down, saving the high fidelity polymerase for last. Run PCR for 10 cycles, then open up PCR tubes, add primers, and continue PCR for 20 more cycles.
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
72
30x
72
12
Time (min.)
2:00
0:30
0:30
3:00
30x
5:00
∞
8/24: Gel, Stitch, PCR
Gel Order: Ladder, Rxn 1 (8/23), Rxn 2 (8/23)
PCR Stitch URA3: see 8/23 rxns 1 and 2 (approx. 5μL of 12.7ng/μL DNA and approx. 5μL of 12.1ng/μL DNA)
PCR URA3 Amplification: see 8/19 Rxns 3 and 4; see also for thermocycler temperature and times
8/25: Gel, PCR
Gel Order: Ladder, 1, 2, 3, 4 (from 8/24)
PCR Master Mix 1:
6μL 1A F
6μL 1A R
6μL HF polymerase
150μL green mix
126μL NF water
PCR Master Mix 2:
6μL 1B F
6μL 1B R
6μL HF polymerase
150μL green mix
126μL NF water
Portion into 49μL portions and add
1μL 1A DNA to each of MM1
1μL 1B DNA to each of MM2
1
2
3
4
5
6
7
Temp. (°C)
95
95
50
72
30x
72
12
Time (min.)
2:00
0:30
0:30
2:00
30x
5:00
∞
8/26: Gel, gBlock Amplification, PCR
Gel 1: Ladder, gblock 1, 1A, 1A, 1A, 1A, blank, 1B
Gel 2: Ladder, gblock 2, 1B, 1B, 1B, 1B
gBlock 1: 1A URA3, 44.2ng/μL → 0.045pmol
gBlock 2: 1B URA3, 87.3ng/μL → 0.088pmol
PCR Amplification of gblocks:
    Mix 1:
150μL Gotag
6μL F and R primers
6μL  HF polymerase
6μL 1A URA3 DNA
126μL NF water
    Mix 2:
150μL Gotag
6μL F and R primers
6μL  HF polymerase
6μL 1b URA3 DNA
126μL NF water
    See 8/25 for PCR steps
8/29: Stitch, PCR Amplification
Stitch:
    Rxn 5:
3μL 1A F
3μL 1B R
9μL 1A URA3 ( @ 66.1ng/μL)
9μL 1B URA3 ( @ 77.8ng/μL)
1μL HF polymerase
25μL green mix
Rxn 6:
3μL 1A F
3μL 1B R
12μL 1A URA3 ( @ 44.2ng/μL)
6μL 1B URA3 ( @ 87.3ng/μL)
1μL HF polymerase
25μL green mix
1
2
3
4
5
6
7
Temp. (°C)
95
95
50
68
30x
68
4
Time (min.)
2:00
0:30
0:30
0:30
30x
10:00
∞
1
2
3
4
5
6
7
Temp. (°C)
95
95
50
68
30x
68
4
Time (min.)
2:00
0:30
0:30
3:10
30x
10:00
∞
PCR Amplification of gBlock URA3 DNA
URA3 1A:
75μL Gotag
3μL F and R primers
3μL  HF polymerase
3μL 1A URA3 DNA
63μL NF water
    URA3 1B:
75μL Gotag
3μL F and R primers
3μL  HF polymerase
3μL 1B URA3 DNA
63μL NF water
Portion into 3 50μL amounts and place in 3 PCR tubes
8/30: Gel
Gel Order: ladder, sarah 5, sarah 6, chase 5, chase 6, 1, 2
PCR Purify: 1 and 2
8/31: Gel
Gel: Ladder, 1.1, 2.1, 3.1, 4.1, 1.2, 2.2, 3.2, 4.2
9/1: Stitch
Mix 1
Mix 2
Mix 3
Mix 4
25 uL GoTaq
25 uL GoTaq
25 uL GoTaq
25 uL GoTaq
1.0 uL 1A DNA
1.0 uL 1A DNA
2.0 uL 1A DNA
2.0 uL 1A DNA
1.0 uL 1B DNA
1.0 uL 1B DNA
2.0 uL 1B DNA
2.0 uL 1B DNA
23 uL NF water
23 uL NF water
21 uL NF water
21 uL NF DNA
PCR Cycle:
Temperature (°C)
95
45
60
72
Steps 2-4
72
10
Time (min)
3:00
0:30
1:00
10:00
30X
10:00
∞
Gel:
1.1
1.2
2.1
2.2
Ladder
3.1
3.2
4.1
4.2
Gel extraction Gel: 450 mg
9/7: Gibson Assembly
Add 10 uL +Ctrl to 10 uL GA 2X Master Mix
Add 10 uL GA Master Mix
Add 2 uL URA3 A
Add 2 uL URA3 B
Rxn 1 (Total 40.4 pmol)
Rxn 2 (total DNA ~0.1 pmol)
Rxn 3
10 uL GA mix
10 uL GA mix
5 uL GA mix
2 uL 1A at 112 ng/uL
5 uL 1A at 11.2 ng/uL
5 uL + ctrl
2 uL 1B at 128 ng/uL
5 uL 1B at 12.8 ng/uL
6 uL water
Total
20 uL
20 uL
10 uL
Incubate at 50° C for 15 minutes.
PCR
Mix A
Mix B
25 uL GoTaq
25 uL GoTaq
1 uL 1AF primer
1 uL 1Af primer
1 uL 1BR primer
1 uL 1BR primer
1 uL DNA (1, 1/10X)
5 uL DNA (1, 1/10X)
1 uL poly (phusion)
1 uL HF poly (phusion)
21 uL water
17 uL water
Total
50 uL
50 uL
PCR Cycle
Temperature (°C)
95
95
60
68 (X30)
68
4
Time (min:sec)
2:00
0:30
0:30
3:10 (X30)
10:00
∞
M. Mix
A
B
125 uL GoTaq
125 uL GoTaq
5 uL 1AF
1 uL 1AF
5 uL 1BR
5 uL 1BR
5 uL poly
5 uL poly
10 uL water
85 uL water
Notes:
J=rxn 1@ 1X
K=rxn 1@ 1/10X
I=rxn 2@1X
L=rxn 2@1/10X
Ja=rxn 1@1X with PCR condition A
9/9
PCR:
25 uL GoTaq
1 uL 1AF
1 uL 1BR
1 Ul HF poly
1 uL DNA
21 uL water
E=1     F=1/10 of 1       G=2         H=1/10 of 2
Temperature (°C)
95
95
55
68 (X30)
68
4
Time (min:sec)
2:00
0:30
0:30
3:10 (X3)
10:00
∞
9/12
PCR:
25 uL GoTaq
125 uL GoTaq
1 uL AF
5 uL AF
1 uL BR
5 uL BR
I uL HF poly
5 uL HF poly
1 uL DNA
-
21 uL water
105 uL water
PCR times:
Temperature (°C)
95
95
60
68
X30
68
4
Time (min:sec)
2:00
0:30
0:30
3:10
X30
10:00
∞
9/12: PCR, Gel
PCR:
25μL Gotag
1μL F and R primers
1μL  HF polymerase
1μL DNA
21μL NF water
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
72
30x
68
4
Time (min.)
2:00
0:30
0:30
3:10
30x
10:00
∞
Gel:
9/13: PCR
PCR:
25μL Gotag
1μL F and R primers
1μL  HF polymerase
1μL DNA
21μL NF water
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
68
30x
68
4
Time (min.)
2:00
0:30
0:30
3:10
30x
10:00
∞
9/14: Gel, Gibson Assembly, PCR
Gel: M, K, O, L
    \* M was dehydraded and resuspended solidified green mixture w/water and loaded
Gibson Assembly: Recovery Drive
    1 @ 100ng/uL → 0.11pmol
1 @ 10ng/uL → 0.011pmol
2 @ 100ng/uL → 0.088pmol
2 @ 10ng/uL → 0.0088pmol
3 @ 100ng/uL → 0.11pmol
3 @ 10ng/uL → 0.011pmol
Rxn A:
5uL green mix
1uL RD 1 @ 100ng
1uL RD 2 @ 100ng
1uL RD 3 @ 100ng
2uL water
Rxn B:
5uL green mix
1uL RD 1 @ 10ng
1uL RD 2 @ 10ng
1uL RD 3 @ 10ng
2uL water
Incubate samples at 50 degrees C for 15 minutes
PCR:
Rxn 1=RD1
Rxn 2=RD2
Rxn 3=RD3
Rxn 4=Rxn A + RD1F and RD3R;
Rxn 5=Rxn B+RD1F and RD3R
25μL Gotag
1μL F and R primers @ 10uM
1μL  HF polymerase
1μL DNA
21μL NF water
1
2
3
4
5
6
7
Temp. (°C)
95
95
56
68
30x
68
4
Time (min.)
2:00
0:30
0:30
2:00
30x
10:00
∞
9/15: Gel, PCR
Gel Order: Ladder, 1, 2, 3, 4, 5
PCR: Set up 2 of reactions 1, 2, and 3 and run each at one of two PCR cycles (A or B)
Rxn 1:
25uL green mix
1uL RD 1
1uL RD1 F
1uL RD1 R
21uL water
1uL HF polymerase
Rxn 2:
25uL green mix
1uL RD 2
1uL RD2 F
1uL RD2 R
21uL water
1 uL HF polymerase
Rxn 3:
25uL green mix
1uL RD3
1uL RD3 R
1uL RD3 F
21uL water
1 uL HF polymerase
Cycle A
1
2
3
4
5
6
7
Temp. (°C)
95
95
58
72
30x
72
10
Time (min.)
2:00
0:30
0:30
1:30
30x
10:00
∞
Cycle B
1
2
3
4
5
6
7
Temp. (°C)
95
95
60
72
30x
72
10
Time (min.)
2:00
0:30
0:30
1:30
30x
10:00
∞
9/16: Gel, PCR
Gel Order: Ladder, 1A, 1B, 2A, 2B, 3A, 3B
PCR:
50uL green mix
2uL RD 1
2uL RD1 F
2uL RD1 R
43uL water
1uL HF polymerase
Total: 100 uL
This mixture was used twice.
PCR:
Temperature °C
95
95
55
72
(previous steps X30)
72
12
Time (min:sec)
2:00
6:30
0:30
2:00
5:00
∞
(I wasn’t really sure what the above table was supposed to say exactly. Kind of hard to read.)
9/19
Extended GA w/ 1+2 RD (w/ gel extract product)
1 uL
RD 1
1 uL
RD 2
3 uL
water
5 uL
GA mix
Incubated at 50° C for 1 hour.
PCR:
50 uL GoTaq
43 uL water
2 uL RDIF
2 uL RD2R
2 uL GA rxn
1 uL Phusion
Temperature (°C)
95
95
60
72
(previous steps X30)
72
12
Time (min:sec)
5:00
0:30
0:30
4:00
5:00
∞
This was done using gradient PCR with gels A-H. Gels E-H had bands.
9/23
Gel of RDI+2
PCR:
50 uL GoTaq
43 uL water
2 uL GA product
2 uL RD2R
2 uL RDIF
2 uL Phusion
Temperature (°C)
95
95
50/60
72
(previous steps X30)
72
12
Time (min:sec)
5:00
0:30
0:30
4:00
5:00
∞
9/26
Gel
RD1+2-3 well
GD1
GD+1GD(50°C)
GD2
GD2+GD2(50°C)
GD2(50°C)
Gradient PCR
100 uL GoTaq
80 uL water
4 uL 1AF
4 uL 1BR
4 uL gBlock 1
2 uL Phusion
Temperature (°C)
95
95
70-50
72
(previous steps X30)
72
12
Time (min:sec)
5:00
0:30
0:30
2:00
5:00
∞
9/27
Gel: Ladder, A, B, C, D (excised), E, F, G, H (excised)
Excised gel in -20° C freezer
9/28
PCR:
50 uL GoTaq
43 uL water
2 uL GDIF @ 10 uM
2 uL GDIR Gibson @ 10 uM
2 uL GD1 @ 10 ng, 1 uL
2 uL GD1
1 uL Phusion
Temperature (°C)
95
95
50
72
(previous steps X30)
72
12
Time (min:sec)
5:00
0:30
0:30
2:00
5:00
∞
2 tubes labeled DGI
Gradient PCR
100 uL GoTaq
6 uL water
4 uL GD2F
4 uL GD2R
4 uL GD2
2 uL Phusion
Temperature (°C)
95
95
50-70
72
(previous steps X30)
72
12
Time (min:sec)
5:00
0:30
0:30
2:00
5:00
∞
Tube
Temperature Annealed At (°C)
A
70
B
68.5
C
65.9
D
62
E
57.5
F
53.8
G
51.4
H
50.0
Ideas:
Gibson Assembly: RD2+RD3
Gradient PCR GA RD1+2
Gibson Assembly RD2+RD3:
1 uL RD2
2 uL RD3
2 uL water
5 uL GA mix
Incubate @ 50°C for 1 hour
Labelled RD23
Gradient PCR RD 1+2
100 uL GoTaq
68 uL water
4 uL RDIF
4 uL RD2R
4 uL RD1+2
2 uL Phusion
Temperature (°C)
95
95
50-70
72
(previous steps X30
72
12
Time (min:sec)
5:00
0:30
0:30
5:00
5:00
∞
Tube
Temperature Annealed At (°C)
J
70.0
K
68.5
L
65.9
M
62.0
N
57.5
O
53.8
P
51.4
Q
50.0
Gradient PCR RD 2+3:
100 uL GoTaq
68 uL water
4 uL RD2F
4 uL RD3R
4 uL RD2+3
2 uL Phusion
Temperature (°C)
95
95
50-70
72
(previous steps X30
72
12
Time (min:sec)
5:00
0:30
0:30
5:00
5:00
∞
Tube
Temperature Annealed At (°C)
S
70.0
T
68.5
U
65.9
V
62.0
W
57.5
X
53.8
Y
51.4
Z
50.0
9/29
Gel 1 Ladder: A B C D E F G H
Gel 2 Ladder: J K L M N O P Q GD1
Gel 3 Ladder: S T U V W X Y Z GD1
Gels 1 and 2 showed no bands at described spots(same for Gel #3)
Nothing was cut from any of the gels.
Ladders A-H:
Ladders J-Q:
Ladders S-Z:
10/3
Gradient PCR
100 uL GoTaq
86 uL water
4 uL GD2F at 10 uM
4 uL GD2R at 10 uM
4 uL GD2 (template)
2 uL Phusion
Temperature (°C)
95
95
50-70
72
(previous steps X30
72
12
Time (min:sec)
5:00
0:30
0:30
2:00
5:00
∞
10/4
Gblock 1: PCR amplification from 7/25 was repeated, marking 2 50-mL samples of GD1 (1A, 1B) and 2 50-mL samples of GD2 (2A, 2B).
RD: Successful procedures from previous days:
Gblock 1: See procedure from 9/14 and 9/15
Gblock 2: See procedure from 9/15
Gblock 3: Couldn’t find successful amplification
10/5
Gel Ladder: 1A, 1A, 1B, 1B, 2A, 2A, 2B, 2B
PCR 1:
50 uL GoTaq
2 uL RD1F
2 uL RD1R
2 uL RD1 @ 10 ng/uL
2 uL HF polymerase
42 uL NF water
PCR 3:
50 uL GoTaq
2 uL RD3F
2 uL RD3R
2 uL RD3 @ 10 ng/uL
2 uL HF polymerase
42 uL NF water
PCR 2:
100 uL GoTaq
86 uL NF water
4 uL RD2F
4 uL RD2R
4 uL RD2
2 uL HF polymerase
Temperature (°C)
95
95
varies\*
72
(previous steps X30)
72
4
Time (min:sec)
2:00
1:00
1:00
3:00
5:00
∞
\*54.7°C for gels 1 and 3; 55°C for gel 2.
Ran gel for DG1 and GD2 PCR. There were no bands. PCR was set up for RD1, RD3, and a gradient of PCR of RD2.
10/6
Gel: Ladder 1 1 3 3
2 bands were present in each of Ladder 1.
Excised and gel extracted 2 bands from 1 labeled 1 1 and stored in -20°C.
10/7
Gradient PCR of RD3:
100 uL GoTaq
4 uL RD3F
4 uL RD3R
4 uL RD3
2 uL HF polymerase
86 uL NF water
Gels 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H were annealed at the same temperature as on 9/28.
Temperature (°C)
95
95
50-70
72
(previous steps X30)
22
12
Time (min:sec)
2:00
1:00
1:00
3:00
5:00
∞
RD3 failed to amplify. Gradient PCR was done to try to see if different annealing temperature will help.
10/10
Gel of gradient PCR RD3
RD1- 54.7°C
RD2- gradient PCR failed
RD3- 50°C
RD3 PCR:
50 uL GoTaq
2 uL RD3F
2 uL RD3R
2 uL RD3
1 uL HF polymerase
45 NF water
Temperature (°C)
95
95
50
72
(previous steps X30)
7
12
Time (min:sec)
2:00
1:00
1:00
3:00
5:00
∞
TetR:
50 uL GoTaq
1 uL TetR F
1 uL TetR R
2 uL RD3
1 uL HF polymerase
45 NF water
Temperature (°C)
95
95
56
72
(previous steps X30)
72
12
Time (min:sec)
2:00
1:00
1:00
3:00
5:00
∞
Gradient PCR RD2:
100 uL GoTaq
4 uL RD2F
4 uL RD2R Gibson
4 uL RD2
2 Ul HF polymerase
86 uL NF polymerase
Temperature (°C)
95
95
50-68
72
(previous steps X30)
72
12
Time (min:sec)
2:00
1:00
1:00
3:00
5:00
∞
ADE2 F:
50 uL GoTaq
1 uL ADE2
2 uL RD1R
2 uL RD1
1 uL HF polymerase
44 uL NF water
PCR performed at same time and temperature as TetR.
ADE2 R:
50 uL GoTaq
1 uL RD2F
2 uL RD2
1 uL HF polymerase
44 uL NF water
PCr performed at same time and temperature as TetR.
PCR RD3 now near temperature
Gradient RD2 with new reverse primer
PCR TetR from D3
PCR ADE2 from RD1
PCR ADE2 R from RD2
10/11
Gel 1 Ladder: A-H
Gel2 Ladder: ADE2F  ADE2F TetR  TetR ADE2R  ADE2R  3  3
2A-H: 1730- fail, band not right
ADE2F: 874- fail, no band
ADE2R: 842- fail, no bands
TetR: 624 bp- okay
RD3: 1378- fail, no band
10/12
Gradient PCR GD1:
100 uL GoTaq
2 uL GD1F @ 100 uM
2 uL GD1F @ 100 uM
4 uL GD1 @ 10 ng/uL
2 uL HF polymerase
Gradient PCR GD2:
100 uL GoTaq
2 uL GD2F @ 100 uM
2 uL GD2F @ 100 uM
4 uL GD2 @ 10 ng/uL
2 uL HF polymerase
Temperature (°C)
95
95
50-70
72
(previous steps X30)
72
12
Time (min:sec)
2:00
1:00
1:00
3:00
5:00
∞
Tubes labelled 1A-H and 2A-H respectively.
Looking for products around 1368bp.
Gel Extract TetR:
Use NEB Monarch Kit gel extraction
Elute using 13 uL of elution buffer
PCR clean-up w? NEB Monarch Kit DNA Clean-up because of accident with CutSmart buffer
Digest:
10uL Tetr
2 uL EcoRI HF
2 uL Pst1 HF
4 uL CutSmart
22 uL water
12 uL pSB1C3
2 uL EcoRI HF
2 uL Pst1 HF
4 uL CutSmart
20 uL water
37°C for 1 hour.
10/14
Gel
Temperature (°C)
GD1A-H
68.5
GD2A-H
50
TetR
redo
PSB1C3
redo
PCR GD1:
50 uL GoTaq at 100 uM
1 uL GD1R at 100 uM
1 uL GD1 at 100 uM
2 uL GD1
1 uL HF polymerase
45 uL water
PCR GD2:
50 uL GoTaq at 100 uM
1 uL GD2R at 100 uM
1 uL GD2 at 100 uM
2 uL GD2
1 uL HF polymerase
45 uL water
TetR:
50 uL GoTaq
1 uL TetR F
1 uL TetR R
2 uL RD3
45 uL water
1 uL HF polymerase
Temperature (°C)
95
95
varies\*
72
(previous steps X30)
72
12
Time (min:sec)
2:00
1:00
1:00
3:00
5:00
∞
\*68.5°C for GD1; 50°C for GD2; 56°C for TetR
Gel extract TetR+PSB1C3 using NEB kit
Ligation:
PSB173: 11.78 pmol
TetR: 157.6 pmol
3:1
5 uL PSB1C3
1 uL TetR
2 uL T4 ligase buffer
1 uL T4 ligase
11 uL water
5:1
2.67 uL PSB1C3
1 uL TetR
2 uL T4 ligase buffer
1 uL T4 ligase
13.33 uL water
10/15
Gel:  GD1, GD2, TetR
Transform UQ950 cells:
Add <20 uL DNA to competent cells
Incubate 30 min on ice
Incubate 2 min @ 42°C
Incubate 1 hr @37°C shaking in 750 uL RB
Plate 1:1
10/16
Pick colonies and start liquid culture.
1 per plate.
4mL LB
4 uL chloramphenical
Put plate back into 37°C.
10/17
Glycerol stock:
1.5 mL E. Coli was spun down to 500 uL
500 uL 50% glycerol
3722- PSB1C3: TetR (1:3 ligation)
3723- PSB1C3: TetR (1:5 ligation)
Miniprep:
Using NEB kit, elute into 30 uL buffer
Gel extract GD1=GD2 from 10/15
Using NEB kit, elute into 17 uL buffer
Restriction digest
16 uL DNA
2 uL CutSmart
1 uL SpeI
1 uL PstI
Incubate at 37°C for 1 hour
Gibson Assembly:
1 uL GD1 at 15.3
4 uL GD2 at 5.82
5 uL Gibson Assembly Mix
Incubate at 50° C for 1 hour.
PCR GD12 Stitch:
100 uL GoTaq
2 uL GD1F
2 Ul GD1R
4 uL Gibson Assembly
2 uL HF polymerase
90 uL water
PCR GD1:
50 uL GoTaq
1 uL GD1F
1 Ul GD1R
2 uL Gibson Assembly
1 uL HF polymerase
45 uL water
PCR GD2:
50 uL GoTaq
1 uL GD2F
1 Ul GD2R
2 uL Gibson Assembly
1 uL HF polymerase
45 uL water
Gel: 120 volts for 30 min
Gel extract: Used NEB kit; elute into 30 uL buffer
10/18
Digest:
1 uL EcoRI HF
1 uL Pst1 HF
11.5 uL DNA (TetR) at 266 ng/uL
2 uL CutSmart
4.5 uL NF water
Total: 20 uL
Gel extract performed
Ligation:
1 uL vector
3 uL insert
2 uL buffer
1 uL T4 ligase
13 uL water
1 uL vector
5 uL insert
2 uL buffer
1 uL T4 ligase
11 uL NF water
(Insert: 22 pmol.   Vector: 18 pmol)

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