Team:Minnesota/Notebook

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Please refer to our notebook document herefor a better formatted version of our notebook with gel images.

>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:

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.

>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)