208 lab

Main purposes today

helloworld-project

• 25 rxns including duplicates

• 34 rxna including duplicates with the following setup:
  • Tube 1-6 containing 5uL pZA21 plasmid template and 3uL of each primers for amplification of the backbone.
  • Tube 7-10; 1uL g-Block from previous purification and 3uL of each Sec signal primers.
  • Tube 11-14 containing same amount of g-Block and primers but now replaced with the TAT2 signal primers.
  • Tube 15-18; 5uL GFP SF plasmid template and 3uL of each primers for GFP SF TAT.
  • Tube 19-22; 5uL GFP SF plasmid template and 3uL of each primers for GFP SF Sec.
  • Tube 23-26; 5uL RFP plasmid template and 3uL of each primer for RFP.
  • Tube 27-30; 1uL g-Block purification and 3uL of each TAT3 primers.
  • Tube 31-34; 0.5uL of the original g-Block and 3uL of each primers for amplifying the g-Block.

All tube with less than 5uL template DNA was filled with MQ until final volume of 11uL(5+3+3).

First half of the samples where run on PCR with x7-polymerase and the second half where run with PHUSION-polymerase, with the exception of the g-Block where all reactions where run with PHUSION. This gives following setup: x7-poly in tube:

• 1-3, 7-8, 11-12, 15-16, 19-20, 23-24, 27-28

PHUSION in tube:

• 4-6, 9-10, 13-14, 17-18, 21-22, 25-26, 29-30, 31-34

A mastermix was made for both PCR with standard concentration setup and one additional volume for pipetting errors:

x7 mastermix for 16rxn

• 160uL HF buffer
• 16uL dNTP's 10mM
• 8uL x7 polymerase
• 440uL MQ

PHUSION mastermix for 20rxn

• 200uL HF buffer
• 20uL dNTP's 10mM
• 10uL x7 polymerase
• 5500uL MQ

The tubes were run on two programs simultaneously both where ramps. First program with the ramp going from 70°C → 60°C and another from 60°C → 50°C. The tubes in first program(70°C → 60°C) where tube 7-10, 15-26 and for second program(60°C → 50°C) tube 1-6, 11-14, 27-34.

USER reaction and transformation of E.coli

USER reaction was performed according to the standard procedure given with USER enzyme and for sample named U2 after standard reaction we additionally heated the sample to 98 degrees for 2 minutes and cooled down gradually to 4 degrees (0.3 C/s).

Chemical transformation According to the iGEM protocol (transformation protocol) with slight changes.

• 100 μl cells + whole user reaction mix (50 μl)
• step 6 : 90 sec (instead of 60 s)at 42 ^oC
• Add 400 μl of SOC media (instead of 200 μl)

Transformants were plated on plates named: USER U, USER U2, Neg., NEG. NO DNA. Plates were incubated overnight at 37 degrees.

19 colonies were chosen to be verified by colony PCR for fragment containing GFP and RFP (expected length about 1550 bp. No positive results were obtained. We will perform once again amplification of all DNA fragments with x7 polymerase and perform USER reaction and transformation again.

Who was in the lab

Henrike, Jakob, Kristian, Gosia

Procedure

PCR for 7 fragments:

Conclusion from today

Inconsistent results from the first PCR where obtained with bands in some of the reactions but not in the duplicates. Even the g-Block was not seen though we have easily amplified that before with very bright bands. Something must have gone wrong and that's why we made the additional 34rxns which will be analyzed on gels tomorrow.

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