Team:KULeuven/12 September 2008

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Lab Work

Last Friday, we had a lot of good news. This time it is bad news :-(

There were no colonies on the plates of three ligations that we electroporated yesterday (J23116+B0032+C0062+B0015, K145253+ K145254 and J23109+J23032+K145001+B0015). We did a PCR test to see whether the other ones were properly ligated. Only one of them seemed alright (K145272).
We miniprepped some cultures: R0040+J23066+J23109+J23032 and K145278.
The end-filling reaction to make J23078 was put on gel. It appeared to be OK.
We made a number of digests:
cut with EcoRI and SpeI -> R0040+J23066+J13109+J23032 (succeeded).

cut with XbaI and EcoR I -> K145278 (failed).

cut with XbaI and Pst I -> J23078 (PCR product).

cut with SpeI and Pst I -> C0062, J23109 and I712074 (J23109 failed).
We set up two more ligations: J23116+B0034 and K145275.
We also continued our quest for T7 polymerase with UmuD tag. It was suggested that the problem may lie in the fact that we use a gel extract as a template (there might still be a lot of agarose in the sample). Therefore we started step 1 of the PCR (first halve of the tag). We will do step 2 of the PCR (second halve of the tag) tomorrow with the unpurified PCR mix of step 1 as template.