Hi everyone, I just started working in a lab for the first time (so please forgive my poor technical language and gaps in knowledge!!), and am having a lot of trouble doing a simple transformation. Each time I've ended up with little to no colonies, with the existing colonies being false-positive. My procedure goes something like this:
Insert: ERCC1 (v1/2/3)
- PCR ERCC1 (40ul of reaction, used this much because I have trouble with low yield in the following purification procedure)
- Gel purification
- Double digest (SalI, BamHI) vector and insert
- Gel purification of pEGFP and column purification of ERCC1
- Ligation (total volume 35ul, used 4ul in transformation)
- 0.2ug of ERCC1 variants (~30ul)
- 0.03ug of vector (1ul)
- T4 ligase (new)
- Transformation (heat shock method)
I know the competent cells work because I ordered it from a manufacturer, and transformation of their control gives me a lawn. Transformation of parent vector also gives me a lawn.
I also began to doubt whether is my own technical issue, and asked a grad student (who's been doing it successfully) to help me transform my ligation mix. And -> no colonies.
So I'm wondering what is up with my ligation mix? I even measured the concentration of DNA using nanodrop and followed the vector:insert ratio according to the 1:3 rule.Can it be possible that I've damaged my DNA from UV exposure? However, each time I ran a gel to check the presence of DNA I get a clear band (definitely some loss there, but the bands are present).
At my wits end! Someone help a newbie out