Difference between revisions of "Team:BroadRun-NorthernVA/Notebook"

Week 2

Gel Electrophoresis
• 10µl of each PCR reaction was added into ten different tubes and 2µl of loading dye added.
• 11µl was then loaded into each of the wells and ran for 15 minutes.
Week 3
PCR Purification
• 30 µl of PCR product out of 100 µl was purified, using a Quiagen PCR purification kit.
Restriction Digest
• Total reaction volume was 50 µl
• Concentrations of plasmids and PCR products
• pSB1c3 25ng/µl
• pRS426 129ng/µl
• pAG36 900ng/µl
• PCR products 100ng/µl
Restriction Digest of Plasmids

pSB1c3 plasmid	pAG36 yeast vector	pRS426 yeast vector
4µl DNA	7.8 µl DNA	1.1 µl DNA
5 µl 10x NEB Cut Smart Buffer	5 µl 10x NEB Cut Smart Buffer	5 µl 10x NEB Cut Smart Buffer
1µl EcoR1 enzyme	1µl Kpn1 enzyme	1µl EcoR1 enzyme
1µl Pst1 enzyme	1µl Spe1 enzyme	1µl Pst1 enzyme
39 µl water	35.2 µl water	41.9 µl water

Restriction Digest of PCR Products

PCR product 1 (promoterless and native secretion sequence), cut with EcoR1 and Pst1	PCR product 2 (cyc promoter and native secretion sequence), cut with EcoR1 and Pst1	PCR product 3 (cyc promoter and mating factor alpha1 secretion sequence), cut with EcoR1 and Pst1	PCR product 3 (cyc promoter and mating factor alpha1 secretion sequence), cut with Kpn1 and Spe1
10 µl DNA	10µl DNA	10µl DNA	10 µl DNA
5 µl 10x NEB Cut Smart Buffer	5 µl 10x NEB Cut Smart Buffer	5 µl 10x NEB Cut Smart Buffer
1µl EcoR1 enzyme	1µl EcoR1 enzyme	1µl EcoR1 enzyme	1µl Kpn1 enzyme
1µl Pst1 enzyme	1µl Pst1 enzyme	1µl Pst1 enzyme	1µl Spe1 enzyme
33 µl water	33 µl water	33 µl water	33 µl water

• After ligation, we completed a bacterial transformation and plated cultures on agar plates.

Week 4

The PCR mix components were combined in a large microcentrifuge tube with:

• 100µl of 2x master mix
• 16µl of VF2 primer
• 13 µl of VR primer
• 71 µl of sterile water
• 20µl of the mix was added into each of nine PCR tubes.

Three colonies were chosen from each of the pSB1c3 ligations that had been transformed into the E.coli, labeled 1A,1B,1C, 2A, so forth. Each colony was mixed into the PCR mix and then streaked onto a master plate.

• 10µl of the finished PCR product was mixed with 2µl of loading dye and loaded into a agarose gel.

  The results confirmed that our insert size was correct

  Next, we mini-prepped our liquid cultures using Quiaprep mini prep kit. There were eighteen cultures total, three colonies for each of the five transformations.

  After the mini prep, we needed to check the size of the DNA and the DNA had not been lost during the miniprep. To linearize the DNA, 5µl was added to 45 µl of a master mix.

  Two master mixes were used, one with EcoR1 and one with Spe1. The ligations with the pSB1C3 plasmid and yeast vector pRS426 contain a single EcoR1 restriction site and the pAG36 yeast vector contains a Spe1 restriction site. Master mix with Ecor1 was made for 15 reactions and the Spe1 master mix for 5 reactions.

  Master mix:

  
  
  

  Ecor1 mix:

• 75 µl 10x New England Bio Labs Cut Smart Buffer
• 15 µl EcoR1 enzyme
• 585 µl distilled water
• Spe1 mix:
• 25 µl 10x New England Bio Labs Cut Smart Buffer
• 5 µl Spe1 enzyme
• 195 µl distilled water

  DNA was incubated at 37° Celsius for one hour.

  10 µl of loading dye was added, the solution was pipetted up and down to mix thoroughly.

  20µl was loaded into each well in addition to a 1kb DNA ladder.

  Week 5

  Master Mix 1:

  Primer 1	Primer 2	std taq	dntps	taq	h2o
  10	10	20	4	2	134

  Master Mix 2:

  Primer 2	Primer 3	std taq
  10	10	20

  colony pcr gel

• 10 fold dilution(2a 2b 3a 3b 4a 4b 5a 5b 6a 6b)
• 100 fold dilution (2a 2b 3a 3b 4a 4b 5a 5b 6a 6b)
• prs426 positive control
• pag36 pc
• psb1c3 nc

  Project Timeline

  Week 1

• Colony PCR on bacterial cultures
• Made liquid cultures of positive colonies
• Miniprepped liquid cultures
  
  
• Transformed DNA samples into S.cerevisiae

  Week 2

• Tested genetically engineered cells for ability to produce amylase and hydrolyze starch
• Cells were tested in known concentrations of soluble starch dissolved in water Iodine was used to determine starch concentrations
• Then the yeast cells were tested in a sample of Armstrong's factory water

Week 3

• Cells were heat killed at 80 degrees Celsius to inactivate enzymes.
• Ran through a gel to verify the fragments
• Ligation of gene after successful gel.

  Week 4

• Today we ran colony PCR to confirm the insert size. Our bacterial transformations went well, and we had both liquid cultures and agar plates with the colonies. The synthesized primers from IDT for the yeast vectors have not arrived, so colony PCR was done only on the three transformations with the pSB1c3 vector.

  Week 5

• Colony PCR
• Starch Analysis
• psb13 plasmid results