Team:WashU/Notebook

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Notebook

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

May 31

• Preliminary Shopping List

1. 1. PCR buffer w/o MgCl - 5ML - $45.00
   2. M8787- 5ML -MgCl Reagent- $49.30
   3. D7295-.5ML - dNTP - $73.00
   4. D4184-250UN - Taq Polymerase - $150.00
   5. D8045-250UN - AccuTaq - $300
   6. E1385-5ML – Ethidium Bromide – $23.70
   7. T8280-1L – Tris Acetate EDTA Buffer – $45.10
   8. NA1020-1KT – PCR Clean-Up Kit -$102.50
   9. NA1111-1KT - Gel Extraction Kit - $102.00

Subtotal: $890.60

• Met with Cohen Lab grad students to finalize plan and get cassettes.
• Received Leu2 cassette in pRS305 plasmid.
• Received Ura3 cassette in pRS306 plasmid.

June 1

• Lesson from Bert Berla on how to design primers
• Codon Optimized the four genes using tool at: http://www.encorbio.com/protocols/Codon.htm
  • Double checked gene and restriction sites to make sure enzyme does not cut in the middle of the gene
• Design Primers for cassettes
  • Ura3, Leu2, KanMX4, and NatMX4

June 2

• Made two liters of LB solution
  • 25g of LB Broth per Liter (10g Tryptone, 5g Yeast Extract, and 10g NaCl)
  • Autoclaved solutions: 30 minute sterilization.
  • Refrigerated after cooling.

June 3

• Made YPD

1. 1. In 800ml water in 1 L bottle dissolve 10g of BactoYeast extract
   2. Dissolve 20g of BactoPeptone in the above solution
   3. In 200ml water in 500ml bottle dissolve 20g Dextrose
   4. Autoclave both, combine after autoclaving. (30 minute sterilization)

• Made 1000X Ampicillin Stock
  • Added 2g Ampicillin to 20ml water.
  • Added NaOH until dissolved.
  • Sterile filtered.
  • Frozen in -20C Freezer

• Made cultures of E. coli strains with KanMx4 and Natmx4 inserts.
  • In culture tubes, added 5ml LB and 5ul 1000X Ampicillin Stock.
  • Transferred one colony per strain from plates to culture tubes.
  • Incubate at 30 degrees Celsius shaking at 225rpm overnight.

• Made cultures of Yeast strains BC177 and BC178
  • Added 5ml YPD to two culture tubes.
  • Transferred one colony per strain from plates to culture tubes.
  • Incubate at 30 degrees Celsius shaking at 225rpm overnight.

June 4

• Making freezer stocks of yeast cultures BC177 and BC178
  • Add 1.25mL glycerol to yeast culture
  • Freeze yeast culture in -80 degree freezer

• Make E.coli mini-preps for plasmids containing KanMX4 and NatMX4 (Sigma-Aldrich Mini-Prep Kit)
  • Transferred 4 ml of each culture to microcentrifuge tubes.
  • Centrifuge for one minute at 12000xg
  • Pour out supernatant
  • add 200uL of resuspension solution with RNase
  • add 200uL of lysis buffer
  • add 350uL of neutralization buffer
  • Centrifuge for 10 minutes at 12000xg
  • Insert a Miniprep Binding Column into microcentrifuge tubes and add 500uL of Column Preparation Solution to each miniprep column.
  • Centrifuge at 12000xg for 1 minute. Discard the flow-through
  • Take the lysis of the cultures resulting from the 10 minute centrifuge and pipet it into the binding column
  • Centrifuge at 12000xg for 1 minute. Discard the flow-through
  • Add 500uL of Wash Solution 1 to the column and centrifuge at 12000xg for one minute. Discard the flow-through.
  • Repeat the previous step
  • Add 750uL of Wash Solution 2 (with ethanol) to the column and centrifuge at 12000xg for one minute. Discard the flow-through.
  • Centrifuge at 12000xg for one minute.
  • Transfer the column to a fresh collection tube and add 100uL of Elution Solution to the column
  • Centrifuge at 12000xg for one minute.
  • Store the eluate at -20 degrees C

• This procedure was done separately for the plasmids that contain KanMX4 and NatMX4.

June 5

Read absorbances for all four solutions that contain plasmids with our cassettes: Ura3, Leu3, KanMX4, and NatMX4.

Data was obtained using a nano-drop spectrophotometer. Absorbance was observed between 250nm and 280nm.

DNA Concentrations were as follows:

• Ura3: Very high absorbance reading, must be diluted 1/10 in order to get accurate concentration reading.
• Leu2: Very high absorbance reading, must be diluted 1/10 in order to get accurate concentration reading.
• KanMX4: 30.5 ng/uL
• NatMX4: Very low absorbance reading, eluent was discarded.
• Last year's NatMX4 solution: Very high absorbance reading, must be diluted 1/10 in order to get accurate concentration reading.

• Made new culture of NatMX4
  • 5ml of TB + 5uL of 1000x Ampicillin
  • Incubated overnight at 31 degrees Celsius.

June 6

• LEU2 group
  • Make dilution for nanodrop
    • Make solution of 5uL LEU2, 45uL DI water
    • Concentration: 42.0 ng/uL

• URA3 group
  • Make dilution for nanodrop
    • Make solution of 5uL URA3, 45uL DI water
    • Concentration: 35.9 ng/uL

• KanMX4 group
  • 30.5 ng/uL (from previous day)

• NatMX4 group
  • Performed mini-prep on previous day's culture
  • 200uL eluent with concentration of 28.7 ng/uL
  • Made a 1/10 dilution of last year's NatMX4 stock. (DNA Concentration: 23.3 ng/ul)

June 7

• Ordered PCR materials and electrophoresis materials from Sigma-Aldrich.
• Designed primers to add homology to left side of cassette and an AvrII site to right side of cassette.
• Primer designs are:

Leu2

• Forward: TTT CCT AGG CCA AAC TGG AAC AAC ACT CAA CCC

• Reverse: ATA TTT AAT TAT TGT ACA TGG ACA TAT CAT ACG TAA TGC TCA ACC TAA TTT CGT GTC GTT TCT ATT ATG AAT TTC ATT TAT

Ura3

• Forward: TTT CCT AGG ACC ACA GCT TTT CAA TTC AAT TCA TCA TTT

• Reverse: AGT ATC ATA CTG TTC GTA TAC ATA CTT ACT GAC ATT CAT AAC CGC ATA GGG TAA TAA CTG ATA TAA TTA AAT TG

KanMX4

• Forward: TTT CCT AGG AGC TTG CCT CGT CCC CGC C

• Reverse:ATG AAC ATA TTC CAT TTT GTA ATT TCG TGT CGT TTC TAT TAT GAA TTT TCG ACA CTG GAT GGC GGC GTT

NatMX4

• Forward: TTT CCT AGG AGC TTG CCT CGT CCC CGC C

• Reverse: GGG TAA TAA CTG ATA TAA TTA AAT TGA AGC TCT AAT TTG TGA GTT TAG TCG ACA CTG GAT GGC GGC GTT

June 8

• Split into four technique groups

• PCR group
  • Practice PCR
    • Mastermix:
      • 10x buffer:10uL
      • betaine: 25 uL
      • dNTP: 2.0uL
      • Primer 1: 2.5 uL
      • Primer 2: 2.5 uL
      • Taq polymerase: 5 uL
      • dH20: 28.0 uL
    • Sample 1:
      • 3uL MgCl2
      • 1uL DNA template
      • 16 uL master mix
    • Sample 2:
      • 4uL MgCl2
      • 1uL DNA template
      • 15 uL master mix
    • Sample 3:
      • 5uL MgCl2
      • 1uL DNA template
      • 14 uL master mix
    • Sample 4 (Negative Control):
      • 4uL MgCl2
      • 15 uL master mix

94 degrees: 4min

34 Cycles:

• 94 degrees: 30 sec
• 58 degrees: 30 sec
• 72 degrees: 2 min (2kb product)

72 degrees: 5min

4 degrees: hold

Gel Team:

• We made a gel to use tomorrow:
  • 1. Added 500 mL 1xTAE and 5g Agarose
  • 2. Microwaved until dissolved completely
  • 3. Allowed to cool until able to handle and added 9 mL ethidium bromide
  • 4. Poured into gel mold and allowed to cool overnight

June 9

Gel Team:

• We ran the DNA the PCR team amplified yesterday
  • filled the electrophoresis chamber with 1xTAE
  • Added 2 mL loading buffer to each sample
  • put ladder and 4 samples into gel
  • ran electrophoresis for ~1h
• We imaged the gel
  • used UV imaging machine to take picture below
  • [[1]]

• The desired band was not visible. We determined the problem was we were testing the wrong template DNA
• We made a new batch of gels using 400mL of water and 3.5g of agarose

PCR team:

• We repeated PCR with new template DNA and freshly ordered supplies

Recipe per sample for running plasmid DNA:

• 2.5uL 10x buffer
• 1.25 uL dNTP
• 1.5 uL forward primer 44
• 1.5 uL reverse primer 43
• 16.5 uL dH20
• 0.25 uL Accutaq LA DNA polyermerase

Mastermix:

• 12.5uL 10x buffer
• 6.25 uL dNTP
• 7.5 uL forward primer 44
• 7.5 uL reverse primer 43
• 82.5 uL dH20
• 1.25 uL LA polyermerase

Sample 1, 2, 3:

• 1.5 uL DNA of plasmid pTCP2031v
• 23.5 uL Mastermix

Sample 4:

• 23.5 uL plasmid Mastermix

Recipe per sample for running genomic DNA:

• 2.5uL 10x buffer
• 1.25 uL dNTP
• 0.5 uL DMSO
• 0.5 uL forward primer 42
• 0.5 uL reverse primer 43
• 17.0 uL dH20
• 0.25 uL Accutaq LA DNA polyermerase

Mastermix:

• 12.5uL 10x buffer
• 6.25 uL dNTP
• 2.5 uL DMSO
• 2.5 uL forward primer 42
• 2.5 uL reverse primer 43
• 85.0 uL dH20
• 1.25 uL Accutaq LA DNA polyermerase

Sample 5, 6, 7:

• 1.5 uL vector DNA
• 23.5 uL Mastermix

Sample 8: 23.5 uL genomic Mastermix

Run same temperatures as before with 34 cycles

June 10

• Ran PCR products on a 1% agarose gel.
• Used UV imaging machine to produce picture below
• [[2]]

All six expected bands were visible.

Performed gel extraction on all six bands using the Sigma Kit

• excised the bands from the gel. The genomic band was too big so we cut it in half
  • vector:.37g
  • genomic1: .20g
  • genomic2: .21g
• Placed gels in separate collection tubes and added 3 gel volumes of Gel Solubilization Soln. Allowed gels to dissolve
• Prepared 3 binding columns by adding 500uL of Column prep soln and centrifuging for 1 min. Discarded eluate
• Added 1 gel volume isopropanol to each collection tube and mixed thoroughly.
• Loaded the gel solution into the binding columns in 700 uL portions. centrifuged for 1 min and discarded eluate
• Added 700uL of Wash Soln to each column, centrifuged 1 min, and discarded eluate
• Centrifuged columns again for 1 min and discarded eluate
• Transfer each binding column to a fresh collection tube. Added 50uL of Elution Solution and incubate 1 min
• centrifuge 1 min. The elution contains the DNA

June 13

• Performed PCR product clean-up using a Sigma Kit.

gel team:

• we did PCR cleanup
  • Added .5 mL of the column prep soln to 2 miniprep column and centrifuged at 12000g for 1 min. Discard eluate.
  • Added 65 uL of Binding soln and 15 uL of each PCR reaction to separate columns and centrifuged for 1 min. Discard eluate
  • Add .5ml of Wash soln to each column and centrifuge 1 min. Discard eluate.
  • Centrifuge additional 2 min. Discard eluate and place binding column into new collection tube
  • Add 50uL elution soln and allow to incubate 1 min
  • centrifuge 1 min. The PCR product is in the eluate

Nano-dropped four samples:

• Gel extraction vector DNA
  • No DNA present; one large peak at 230nm signifying the presence of protein.
• Gel extraction genomic DNA
  • No DNA present; one large peak at 230nm signifying the presence of protein.
• PCR product clean-up vector DNA
  • 50uL of solution at 36.9ng/uL
• PCR product clean-up genomic DNA
  • 50uL of solution at 18.4ng/ul

June 14

Transformation:

• Made plates with LB agar
  • Added ~300 mL H2O to graduated cylinder
  • Added 12.5 g LB broth, 7.5 g Bacto Agar, and aforementioned water to Erlenmeyer flask
  • Stirred on hot plate to boiling
  • Added water until total volume was 500 mL
  • Autoclaved
  • Returned to stir plate for 15 minutes to cool
  • Added 0.05 g ampicillin (for antibiotic resistance marker) to flask
  • Poured into 20 plates
    • 3 plates discarded because the agar had solidified by the time they were poured

Gel:

• Did a restriction digest
  • 5 uL 10X Buffer (#2 NEB)
  • 30 uL DNA from PCR cleanup of vector DNA
  • 15 uL water
  • 1 uL 1 ku EcoRI
  • incubated at 37 C overnight

June 15

Assay Preparation:

• Make 500ml YPD
• Autoclaved YPD and poured into 16 petri dishes

June 16

Biobrick Transformation in E. coli:

• GC5 E. coli cells and SOC medium arrived
• Began transformation of E. coli with Leu2 and Ura3 biobricks
  • Thawed 50 uL cells in ice bath
  • Added 10 uL sterile ddH2O into biobrick wells 11A and 11C in plate 3
  • Waited 5 minutes for DNA to resuspend
  • Transferred the 10 uL to microcentrifuge tubes
  • Added 20 uL cells to Ura3 tube, 20 uL cells to Leu2 tube, 10 uL cells to control tube
  • Added 2 uL biobrick DNA to respective tubes
  • Waited 30 minutes for cells to transform on ice
  • Heat shocked in water bath at 37 C for 45 seconds
  • Placed cells on ice for 2 minutes
  • Added 200 uL SOC medium into each tube (100 uL into control tube)
  • Put tubes in shaker at 37 C at 220 rpm for one hour
  • Spread onto plates with LB agar (made June 14)
    • Using a total of 6 plates, spread 50 uL onto one plate and the rest (172 uL) onto the other, for each of the three tubes

Gel:

• Ran digestion on a gel
  • [3]
• Ran a new digestion
  • 30 uL DNA from PCR cleanup of genomic DNA
  • incubated at 37 C overnight

Assay Preparation:

• Streak yeast strains, BC177 and BC178, each onto a separate petri dish with YPD

June 17

Tranformation:

• Due to the presence of ampicillin on the plates, we expected no colonies on the control plates, but colonies should grow on the Leu2 and Ura3 plates because they should have antibiotic resistance against ampicillin, if the transformation succeeded. Colonies grew on all plates, even the negative controls; we assume it must have been caused by deficient or faulty amipicillin. Because of this, we cannot determine whether the transformation proceeded correctly or not.

• We are performing a test of our amicillin stock as well as our new ampicillin stock.
  • Three culture tubes each with 4 mL of LB. One tube is a positive control (No AMP), one has 4 uL of our old AMP stock, and one has 4 uL of our new AMP stock.
  • Cells that do not contain a plasmid are placed in each culture tube.
  • Began shaking at 240rpm at 37 degrees Celsius at 10:30 am.
  • At 2:30 pm (4 hour incubation) the OD of the positive control was around .250 and both AMP cultures were still around zero.
  • CONCLUSION: Ampicillin stocks are viable. Perhaps the reason for the lawns on the negative control transformation plates were due to Ampicillin being added while the media was still too hot.

• We will be making new plates with new ampicillin, following the same procedures as described before:
  • Made plates with LB agar
    • Added ~300 mL H2O to graduated cylinder
    • Added 12.5 g LB broth, 7.5 g Bacto Agar, and aforementioned water to Erlenmeyer flask
    • Stirred on hot plate to boiling
    • Added water until total volume was 500 mL
    • Autoclaved
    • Waited for the solution to cool
    • Added 500 uL ampicillin to flask
    • Poured into 12 plates

Gel

• ran practice digest 2 on a gel
  • [[4]]

PCR Team

• We ran PCR on genomic DNA using two different protocols (Sigma Aldrich's & Bert's) to create more product for the gel team to practice on.

Total samples: 8

Sigma's Protocol:

Recipe per sample for running genomic DNA:

• 2.5uL 10x buffer
• 1.25 uL dNTP
• 0.5 uL DMSO
• 0.5 uL forward primer 42
• 0.5 uL reverse primer 43
• 17.0 uL dH20
• 0.25 uL Accutaq LA DNA polyermerase

Mastermix:

• 12.5uL 10x buffer
• 6.25 uL dNTP
• 2.5 uL DMSO
• 2.5 uL forward primer 42
• 2.5 uL reverse primer 43
• 85.0 uL dH20
• 1.25 uL Accutaq LA DNA polymerase

Sample 1, 2, 3 (not marked):

• 1.5 uL genomic DNA
• 23.5 uL Mastermix

Sample 4(negative sign in circle): 23.5 uL Mastermix

Bert's Protocol (w/adjustments by May):

Recipe per sample for running genomic DNA:

• 2 uL 10x Buffer
• 0.4 uL dNTP
• 0.5 uL forward primer 42
• 0.5 uL reverse primer 43
• 10.35 uL dH2O
• 0.25 uL Accutaq LA DNA polymerase
• 5 uL betaine

Mastermix:

• 10 uL 10x Buffer
• 2 uL dNTP
• 2.5 uL forward primer 42
• 2.5 uL reverse primer 43
• 51.75 uL dH2O
• 1.25 uL Accutaq LA DNA polymerase
• 25 uL betaine

Sample 5,6,7 (marked with a dot):

• 1 uL genomic DNA
• 19 uL Mastermix

Sample 8(negative sign in circle): 19 uL genomic Mastermix

Anneal at 58 degrees with 34 cycles

Assay Preparation:

• Check growth of yeast strains on petri dishes streaked yesterday.
  • Concluded colonies on plates were too close together
  • Re-streaked two new petri dishes of BC177 and BC178 yeast strains using colonies from yesterday’s petri dishes.

June 18

• Check growth of yeast strains on petri dishes
  • Concluded colonies on plates were too close together for plate with strain BC177.
  • Re-streaked one new petri dish for BC177

June 20

• Check growth of yeast strains on petri dishes
  • Concluded colonies on our new stock plates were isolated enough for streaking
• Isolated one colony from BC177 and BC178 yeast strains and cultured them in test tubes with 5 mL of YPD solution

Tranformation again:

• Thawed 50 uL cells in ice bath
• Added 20 uL cells to Ura3 tube, 20 uL cells to Leu2 tube, 10 uL cells to control tube
• Added 2 uL biobrick DNA to respective tubes
• Waited 30 minutes for cells to transform on ice
• Heat shocked in water bath at 37 C for 45 seconds
• Placed cells on ice for 2 minutes
• Added 200 uL SOC medium into each tube (100 uL into control tube)
• Put tubes in shaker at 37 C at 220 rpm for one hour
• Spread onto plates with LB agar (made June 17)
  • Using a total of 6 plates, spread 50 uL onto one plate and the rest (172 uL) onto the other, for each of the three tubes

Gel Team:

• Did PCR cleanup on PCR product from 6/17
• ran a gel with PCR product from 6/17
  • [[5]]
• ran a digestion of PCR product from 6/17 with .3 uL EcoRI
• ran a gel of the digestion
  • [[6]]
• Did gel extraction of PCR product from 6/17

PCR Team:

Ran PCR on the four cassettes with the newly arrived primers. This is amplify the cassettes, add homology to the insertion site, and add a restriction site.