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selecting recombinants


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#1 philman

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Posted 09 September 2010 - 04:08 AM

Right I did my digest, gel purification, ligation and transformation yesterday. But now I am not sure about the state of recombinants.

My plates are:
Vector alone with Ligase: 100ul - 100 colonies
Vector plus insert 1: 50ul - 64 colonies
Vector plusinsert2: 5 ul - 48 colonies

Uncut vector: far far too many colonies to count


The cut vector was treated with TSAP Alkaline Phosphotase after digestion before gel purification.

My main worry is the low number of colonies in the vector + insert plates when compared to the Vector alone plates, does this imply very low recombination frequency? and how many minipreps should I do to try and ensure I get the insert? If I should do any at all and not just start again, or try the ligation again with higher concentration of insert. My suspicion is that my insert is at a lower concentration to what I thought it was.

Also I know i did not do a control without ligase, that was a mistake I know

#2 philman

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Posted 10 September 2010 - 01:47 AM

I did a control transformation over last night wth just the cut vector with no ligase or insert, and got 120 colonies, so I am guessing that the many colonies I got were due to an incomplete digestion by the enzyme, and then contamination of the sample with those as the gel purification obviously didn't work.

but I am worried that there were no more colonies on the sample plate than on the control, I would have expected at least some of the vector and inserts to ligate surely? or perhaps just too few to see.

I am going to start again from scratch now, running the digestion for much longer (perhaps 4 hours instead of 2 hours) and when doing gel purification run it for longer to ensure any unut bands are better seperated, and then running ligation ovrnight at 4 degrees rather then the 3 hours at room temperature I did this time.

Anyway this is all I can think of to try and solve this, I am worried about my insert though, it is a PCR product PCRed up to have BclI sites at either end with a 4bp extra on the end to ensure the enzyme will cut, then trying to ligate into a vector cut with BamHI. perhas the BclI is not cutting properly, hmm




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