IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/28: Difference between revisions

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*The undigested B15 in lane 6 appears as two bands on account of supercoiling of the B15 plasmid.
*Bands in lanes 2 - 5 were cut out and purified using a Qiagen Gel Purification kit
**The gel purification was done with 300 μL Buffer PB and 400 μL Buffer QG


==Team Genetic Fence==
==Team Genetic Fence==

Revision as of 14:50, 28 June 2010

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Team Allergy

PCR Reactions

Mix of Parts 1,2,3 w/ primers A&B (simultaneous PCR)

Annealing Temp: 60C Extension Time: 15 sec

3 reactions (GFP; Bet; LTP)

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
DNA (Parts "1" "2" "3") 1(parts 1&2); .5 (part 3)= 2.5 total
AB primer mix (1x) 2.5
Water 35


2 reactions (GFP, LTP)

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
DNA (Parts "1" "2" "3") 1(each)= 3 total
AB primer mix (1x) 2.5
Water 35


Step 1: Mix of Parts 1&2 w/ Primers A&2

3 reactions (GFP; Bet; LTP)

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
DNA (Parts "1" "2") 1(each)= 2 total
A2 primer mix (1x) 2.5
Water 35.5


Step 2: Mix of Parts 1&2 & 3 w/ Primers A&B

Annealing temp: Extension temp:

"3 reactions"

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
DNA (Parts "1&2" "3") x(parts 1&2); x(part 3)= y total
AB primer mix (1x) 2.5
Water z


2 reactions (LTP; GFP)

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
DNA (Parts "1&2" "3") x(parts 1&2); .5(part 3)= y total
AB primer mix (1x) 2.5
Water z

Gels

1 (ladder); 2(LTP 1+2); 3 (Bet 1+2); 4(GFP 1+2); 5(Ladder); 6(LTP 1,2,3); 7(Be 1,2,3); 8(GFP 1,2,3); 9(LTP 1,2,3(using .5ul)); 10(GFP 1,2,3 (using .5 uL))

Lanes 2-4 should be ~a little less than 300 bp and 6-10 should be ~450bp

Team Flavour

Miniprep of Miraculin and Brazzein Constructs

  • Protocol used was from the Qiagen Miniprep Kit
Nanodrop Specs
Name Concentration (ng/μL)
Miraculin 1 159.6
Miraculin 2 182.2
Brazzein 1 91.2
Brazzein 2 33.7

DTL (Digestion, Ligation, Transformation) of The Big Three (pENTCUP2, NOSt, NOSt + STOP)

Digestion Reactions
pENTCUP2 NOSt NOSt+STOP B15
DNA 4 7 12 7
NEB Buffer 3 (10x) 2 2 2 2
diH2O 10 7 2 7
Xba1 1 1 1 1
Pst1 1 1 1 1
BSA (10x) 2 2 2 2
  • Digestions were left for 1:30 at 37°C
  • 2.2 μL of DNA Loading Buffer were added to each reaction and loaded onto a 1% Agarose gel (TAE buffer). Gel was ran at 125 V for 30 min.

Digest Gel

Lane Contents
Lane 1 1KB Plus Ladder
Lane 2 pENTCUP2
Lane 3 NOSt
Lane 4 NOSt+STOP
Lane 5 B15 digested
Lane 6 B15 undigested


  • The undigested B15 in lane 6 appears as two bands on account of supercoiling of the B15 plasmid.
  • Bands in lanes 2 - 5 were cut out and purified using a Qiagen Gel Purification kit
    • The gel purification was done with 300 μL Buffer PB and 400 μL Buffer QG

Team Genetic Fence

B21 Innoculation

Inoculated 3 colonies of B21 transformed E.Coli and set them to shake for 8 hours. We will maxiprep the B21 plasmid for use as a vector backbone.

PCR of Gal4DBD and Barnase, attempt 2

Ran 3 tubes of each for a total of 6. 7x Mastermix:

  • 14μL DNTP
  • 3.5μL Polymerase
  • 28μL 5x Buffer
  • 73.5μL DH2O

Each tube contained:

  • 1μL Fwd primer
  • 1μL Rev Primer
  • 1μL Minipreped sample
  • 17μL Mastermix

Reduced annealing temp to 50°C from 56°C which was the last attempt, ran otherwise identical LACIN program

Gel of Gal4 DBD and Barnase from second PCR attempt

Ran 1.2% E-gel of the 6 PCR product tubes.

1 2 3 4 5 6 7 8 9 10 11 12
Ladder Gal4 DBD 1 Gal4 DBD 2 Gal4 DBD 3 Barnase 1 Barnase 2 Barnase 3 Ladder


QIAQuick Purification of PCR Product

Because colonies 1 and 2 of Barstar worked properly on last week's gel, the PCR products in those two tubes were combined and purified using the Qiagen PCR purification protocol.

  • as there was about 25μL of PCR product in the two together, 125μL of PB buffer was added (5x the concentration of the PCR product)
  • pipetted the mix into a QIAquick spin column, spun for 30 seconds at 13,000 rpm
  • discarded flow-through
  • added .75mL PE buffer to the column, spun for 30 seconds
  • discarded flow-through, and spun again for 1 minute
  • moved the column to a fresh eppendorf tube
  • added 50 μL EB buffer to the column, spun for 1 minute
  • labeled the eppendorf tube 'Barstar PCR purified'




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