Cloning Problem ! - (Feb/16/2008 )
I am cutting my vector and insert with KpnI and XhoI but this is the fourth time I got my vector religated, also there are lot of colonies in vector with ligase plate but no
colonies in vector without ligase.
Vector is 3469 bp and Insert is 2554 bp (PCR product that has been cut at ends to make it sticky ends)
I am doing sequential digest and I have tried ethanol precipitation as well as column purification in between the Digest 1 (with KpnI) and digest 2 (XhoI)
Finally I gel purify my vector as well as insert.
Can anyone help me please ?
This sounds as if one of the two enzymes is failing it cut. How far apart are the cut sites in the vector? If they are too close, the second one will fail to cut. Are you sure both enzymes are cutting? Try cutting with the enzymes one at a time and running a gel. You can also ligate your cut vector only and then recut with each of the two enzymes. You should get double-length vector fragments in each case. Single length or high molecular weight fragments indicate a problem.
Thanks for the suggestion, sounds good but here are few more things that I have already tried.
My enzymes are in MCS so they are 18 bp apart, I hope that is good enough.
Also, I have tried cutting the vector individually with each enzyme, and the cut looks good with nice band on the gel. I have also tested both enzyme sites with double digest so as to check if both KpnI and Xho I sites are working fine and got right product sizes on the gel.
Another thing that I tried was use very low amount of vector to start with and digesting for 4 hrs each to make sure each digestion was complete.
I didn't get the part where you said "Single length or high molecular weight fragments indicate a problem", if I am ligating two vectors by say KpnI site then I will get a double length vector fragment. Can you explain please.
also I am using enzymes from Promega, I did doubt that there might be something wrong with enzymes but no, double digest proves that enzymes are good too. I never had such a weired problem.
If you have vector cut with both enzyme and ligate it, you end up with back-to-back vector fragments assembling into high molecular weight complexes (unless they recircularize as pairs). If you then cut either of those complexes with one or the other enzyme, you get double length back-to-back vector fragments. This tells you that the cut site you did not cut was successfully cut and religated. If you get single length fragments when doing this, it tells you that the other enzyme either did not cut, or that it did not religate, both of which are bad. So if you examine the ratio of double to single length fragments when you religate and cut with one of the enzymes, it tells you how well the other enzyme cuts and religates. You need to heat kill the ligase before running the gel (and not use the "quick ligase" kits with PEG in the buffers).
Thanks, I will try this, however I need to get my clone now, its been too many tries. Let me know if I can do anything else to get around this problem, since I have already got the primers with these enzyme sites at the ends it might be difficult for me to change the enzyme sites (about 20 primers, 10 set Rev and Fwd).
and just checking how many bp of skirting do you have around your restriction sites? NEB technical reference has a lisit showing the minimum number of bp needed to skirt a site before it is efficiently digest by a restriction enzyme
As I said in my previous post, there are 18 bp between the two enzyme sites, so 18 bp is the skirt. The maximum bp required are 4 as per the NEB chart, so that doesn't look like its a problem. Although I was able to get my clone 3 days back but had to do a 3 fragment cloning.
thanks for the suggestion though.