EPF-Lausanne/25 August 2009

  

25 August 2009

Miniprep:
Made according to the protocol of Aisgen Miniprep Kit Elution in 50 ul. The RD2 #5 plasmids prep as some that have already been prepared will be sent for sequencing this afternoon.

Mediums
New medium were made to grow the double-transformed DH5 a (LovTap BB and RO2) -> should be without thy.

M9 was redone, pH adjusted with amino acid and filtred. for 1L:
 * 5g glucose
 * 6g Na2PO4
 * 3g KH2PO4
 * 1g NH4Cl
 * 0.5g NaCl
 * 0.12g MgSO4
 * 0.01g CaCl2

Trp Operon synthesis
Using the Klenow fragments protocol and primers:
 * Trp operon Rev
 * Trp operon Fwd

The primers volumes were directly taken from the original primer dilution (iGEM 09 primers box)

1. Running Klenow 1 file on thermal cycler to denature and slowlyy anneal primers (self-annealing) 2. Running Klenow file after adding the Klenow enzyme (for extension)

DNA preparation
For sending to sequencing

Note : BBx means LovTAP biobrick. We identified 7 clones that worked, so x is the number of the clone. RO2#x : we have 4 clones that worked for the read out 2 but the RO2#8 seems to have a weird behaviour even if we know it has the insert, so we didn't use it afterwards.

Double transformation
We made a double transformation of RO2#4/BB1, RO2#5/BB3, RO2#8/BB5 and RO2#10/BB6.

We had each time a total volume of 10ul of DNA, with the same amount of each DNA. The standard protocol was used and the cells were then spread over plates containing Kana + Amp as antibiotics.

People in the lab
Basile, Rafael, Nicolas

 