PCR cloning problem - No colonies - (Feb/05/2006 )

My vector is pET32a (5.9 kb), and the insert is PCR product (about 1.2 kb). Their bands are very sharp on the DNA gel. Then, I use EcoR I and Sac I severally to digest the insert and vector. Finally, I do the gel elute to recover them. Their DNA are about 20 ng/μl. The condition of this ligation is 14℃, overnight. I think the produce of the transformation is no problem. But, when I plate out the ligation product in 37℃, overnight; I don't get any colony. I use 2 monthes to do cloning, and get nothing. I hope someone can help me to find the problem. Thank you!!

How close are the restriction sites in the vector? If they are close, then when one enzyme has cut, you only have a small amount of DNA between the end of the DNA fragment and the restriction site. Restriction enzymes don't digest well unless they have long segments of DNA on both sides. This may also be a problem with your PCR product. The best way to solve this issue is to clone the PCR product into a commercial T-vector and then cut it out. You can be sure then, that your insert has been digested with the correct over-hangs on each side. Go to the NEB manual, at the back it will tell you how many base pairs are necessary on either side of the restriction site for each enzyme to get efficient cutting. With respect to your vector, digest with the enzyme that requires the most DNA on each side of the site, in this case I think its SacI. Digest for 2-4 hours, then add the second enzyme, EcoRI and digest overnight. It will not guarantee total digestion but I have had success using this method. I am almost certain this is your problem, it also comes up a lot in this forum.

Also, are you doing a double digest? Is the buffer compatible for both enzymes?

i also think that sites of the enzymes may be the problem....also others things i can think about....how much of vector are you using for ligation? what is the insert/vector ratio? maybe you just have to increase it...and how much of the mixture are you plating?.....and the most important one...are your cells competant? do you have positive control to confirm it?

To ML1975:
I guess so. Because I read the sequence map of pET32a plasmid again, I find the sites of EcoRI and ScaI are spaced out 2 bases. Is NEB manual called " New England Biolabs"? Someone telled me EcoRI can't catalyze for long time, otherwise it would have nonspecific cutting, is it right? I don't do a double digest, I use the restriction enzymes one by one.


To Kathy:
For ligation, I use 4 μl vector and 4μl insert. The ratio is 1:1. I have 10 μl ligation mixture, and I put all to 100 μl competent cell (not commercial). Then added 100 μl LB (no ampicillin) to shock and incubate in 37℃, so the total volumn is 210 μl. I plated all to the LB/Amp plate. I don't understand the last sentence. What is the positive control?


To roso 9999 98:
Yes, I did. When I finished the PCR, I use the PCR clean kit to purify my PCR product.

Anyway, thank you for your help.......

1:1 ratio is only true if your vector size and your insert sizes are the same....otherwise you have to calculate molar ratio and i suggest you increase it to 1:3 or even 1:10 vector:insert. you should always use positive control like a known vector....the one that hasnt been digested to see if it will be transformed into E coli....if not your cells might not be compitant...

Hi,

2 base pairs is way too close. I am pretty sure that neither enzyme will digest very well (if at all) with only 1-2 basepairs on the side of a restriction site. You can double check this with the NEB catalogue (which I can't find at the moment!), and yes NEB stands for New England Biolabs. I think you will have to use enzymes that are futher apart. Or, clone into just one site and screen. If you dephosphorylate your vector with SAP or Antarctic phosphatase, your odds should be pretty good. Screening at least 10 will give a positive clone. Anyway, the strategy is up to you but my feeling is that this is where your problem lies. Good luck.

M.

I hope that someone can help me. I am trying to clone a 1.7kb fragment into NEB K. Lactis pklac yeast vector (9091bp). I am using the ezymes Xho1 and Sal1. I am removing a small portion of the vector (51bp), so I can see the problem with the cutting efficiency due to enzyme competition. I have already tried adding the second enzyme for overnight digestion and I have not had any success. I also cloned my insert into topo and performed a double digest and gel extraction. Please tell me what else I can do. I feel as though something is going wrong in my ligation step. Do you heat inactivate after ligation? Please let me know what you think.

I hope that someone can help me. I am trying to clone a 1.7kb fragment into NEB K. Lactis pklac yeast vector (9091bp). I am using the ezymes Xho1 and Sal1. I am removing a small portion of the vector (51bp), so I can see the problem with the cutting efficiency due to enzyme competition. I have already tried adding the second enzyme for overnight digestion and I have not had any success. I also cloned my insert into topo and performed a double digest and gel extraction. Please tell me what else I can do. I feel as though something is going wrong in my ligation step. Do you heat inactivate after ligation? Please let me know what you think.