From 2013.igem.org

Revision as of 08:45, 27 June 2013

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.

Who was in the lab

Henrike, Jakob, Kristian

Procedure

PCR for 7 fragments:

Conclusion from today