Random DNA library problem - repair, ligation, digestion problem? TOPO and Zero Blunt (Jul/15/2008 )
1. I am attempting to create a random DNA library. DNA was sheared with a needle, and repaired with T4 Pol. First vector I tried was TOPO (pic#1). This is a pic of DNA extracted from colonies grown on amp plates, and digested with ecor1 to check insert size. TOPO is 4.0kb, the ladder in the gel is Kb+. Which lanes contain inserts in your opinion?
2. I did not seem to get a lot of clones with inserts so I tried Zero Blunt which uses a 1hour 16 C T4 Ligase step. Colonies are gown on kan plates and since TOP10 cells are used, no xgal is needed, it says anyway. My first few plates had blue and white colonies, other ligations/transformations achieved plates with all white colonies. DNA was extracted from white colonies, digested for insert size again but for only 4hs at 37 C (Pic#2). The vector is Zero Blunt, 3.5Kb, the ladder is Kb+. Why is there a ladder pattern? Were these clones under-digested? Where are my inserts?
Any help would be greatly appreciated. I didn't think making a random library would be this diffiuclt.
What are you cutting with? Is it possible that your restriction enzymes are cutting your random inserts?
You could try colony PCR with flanking primers; you would want 1 band per colony, of different sizes for each random insert. This approach might give you a more clear cut answer, and then your PCR products could be later sequenced if you ever needed it.
They are cut with EcoRI which flanks the insert site. I have tried other cut sites and get a similar pattern.
Check your EcoRI digestion protocol . I suspect star activity due to EcoRI.
These results are precisely what you would expect. You prepared your DNA with shearing, then ligated it into a vector. You then cut with EcoRI. This enzyme will cut not just at the cloning site of the vector, but also at all of the EcoRI sites in your insert. You get multiple bands because there should be multiple bands.
Agreed. This is what I was trying to articulate in my previous post. Check your ligations by PCR and look for one amplified band per colony (of different sizes).
But if this pattern is created by EcoRI cutting my insert, why would most of the ladder patterns be the same? They all can't have the same insert.
The image you linked on the Left above looks like there's plenty of variability in the digest pattern from one lane to the next. If you are not sure, check by colony pcr and compare amplicon sizes from different clones. Or, is it possible you have contamination somewhere? In your enzyme stocks?