need help in molecular cloning - molecular biology (Oct/05/2004 )
[COLOR=red]Hi, I am doing the molecular cloning now and I got a very weird result, does anyone can help me? I got my gene from the yeast genome by PCR, and cut with BamH1 and Sal I, also I cut with my vector with the same enzyme, after ligation, I transformed both of my ligation sample and the backbone, but now I found lots of colnies (150) in my backbone plate but nothing in my ligation plate, it's weird to me, does anyone have the same problem before or have some suggestions, any message if appreciated!
Could you give more details as to the vector size, insert size, purification methods, estimation of DNA amounts, etc ?
That would give some hints...
There could be several reasons for your failed cloning. But, first make sure your dual digestion of the vector and /insert is complete. To me, it seems that the vector was not cut completely by either Sal1 or BamHI (reason for colonies on your vector only plate). Also, if you have modified the insert ends by adding the restriction sites by PCR followed by digestion, make sure you have added the needed extra bases (preceeding the actual sequence for the restriction enzyme) at the 5' end of the primers. Most common error made when dealing with digestion of oligomers is under-digestion. For efficient ligations, it is recommended that the modified PCR amplicons be digested for atleast 20 hrs with the respective enzymes. I have had much better luck following this.
For now, follow these simple rules see how it goes.
try and de-phosphorylate your vector this will cut down on religation without the insert.
One thing to watch out for is carrying over the Taq from the PCR into your RE digestion. Taq can be very hard to get off the DNA. With the trace levels of dNTPs present it can fill in the ends of your RE sites. I have had a lot of success Proteinase K treating my PCR fragments before RE digestion to avoid this problem.
DNA sequencing primers
And then we are left with proteinase-K to chew up our restriction enzymes?
How is the prot-K removed?
1) Prot. K self digests so leave for 1 hr at 55C (simple)
2) If you are really worried then phenol extract or column purify your PCR product. The simplest way is just add Prot. K to your PCR tube after it finishes, let it sit at 55C for 1 hr, then clean-up as per normal.
Improving DNA sequencing traces