EPF-Lausanne/29 August 2009

Wet Lab

Glycerol stock of the following clones :

-BB5-RO2#5, clone #1 ("double" construction)

-BB5-RO2#5, clone #4 ("double" construction)

-RO2#4 + BB1, clone #3 (double transformation)

-RO2#10 + BB6, clone #8 (double transformation)

They were put in the -80°C freezer (2nd box).

Although so far the colony PCR on these RO + BB didn't show anything concluding, the fact that they grew in antibiotic selection seems to show that it actually worked.

We also re-plated the double transformation clones on newly made plates containing both AMP and KANA. It grew, so it seems to indicate that what we have is right.

We then did a miniprep of the same clones with the kit.

The following clones had also been cultivated overnight in LB :

-RO2#4 + BB1 clone 1

-RO2#5 + BB3 clone 3

-RO2#8 + BB5 clone 6

-RO2#4 + BB1 clone 3 -> red

-RO2#10 + BB6 clone 3

-RO2#8 + BB5 clone 2

-RO2#10 + BB6 clone 8 -> red

-RO2#5 + BB3 clone 2

They were all LB colour except the two red. All have been induced with a spatule of TRY to see if they would change colour.

We also did a PCR with the Taq platinium protocol, for RO2#5-BB5 (c1), RO2#4-BB1 (c3), RO2#10-BB6 (c2) and RO2#5-BB5 (c4). We changed the protocol in the following way : 1.3 ul of dNTP has been added (because of increased length of the products) and 3 min elongation step has been programmed (same reason). We also used 2 positive controls : BB5 and RO2#8.

Digestion with SpeI and PstI of the 4 clones plus the two controls.

Supposed length and bands:

-For the double transformation : PCR 1100bp BB LovTAP and 1800bp RO2 -> 2 bands, SpeI 1 band for LovTAP (4300 bp) and 2 bands for RO2 (1700 and 2100 bp), PstI 3 bands for LovTAP (257, 446, 3500 bp) 1 band for RO2 (3900 bp)

-For the double construct : PCR 2900 bp, SpeI 2 bands (3254, 1750 bp), PstI 3 bands (2450, 257, 2220 bp).

People in the lab

Basile (Saturday !)