Problems with cloning - (Jun/05/2007 )
I am trying to clone a PCR product (1.2 kb) into pcDNA 3.1 hygro vector. I digest both the vector and PCR product with NheI and XhoI, do a gel purification and set up an overnight ligation. I have got absolutely no colonies for the last 5 attempts that I have done. Every thing else is OK - the quality and quantity of vector and insert used for ligation, quality of water used for cloning, efficiency of commercially available competent cells. Even self ligation, (Xho I cut vector and religated) has not worked. The only thing that works well is the positive control for the transformation. When I spoke to few others they suggested that one should avoid gel purification as quality of agarose affects cloning efficiency seriously. Please help me with any suggestions as I am really desperate...
Have you tried just using a column to clean up the digested vector and PCR product, then going into the ligation? I don't know why but for me this always works better than using a gel extraction kit.
I believe we should start here.
I assume that you are certain that this vector DNA is indeed your vector backbone and not some strange random bit of DNA. Has it been check on gel to make sure the fragment is indeed the vector?
As the vector is not self ligating, 2 potential problem areas come to mind,
1- over dephosphorylation of the vector, resulting in end damage which prevents ligation (looking at the amount of time and quantity of CIP)
2- either your ligase enzyme or ligase buffer has gone bad. The ligase enzyme in particular goes bad quite readily.
over dephosphorylated DNA can be tested by a PNK (polynucleotide kinase) test. Add PNK into the ligation mix. Although looking from your post, it seems that you have not conducted a dephosphorylation on your vector.
This leaves (2), ligase/ligase buffer problems. Has anyone in your lab conducted a sucessful ligation recently? How old is the lab's T4 ligase enzyme? Try to borrow some ligase and ligase buffer that is known to work. If unable, consider buying some new T4 ligase. And remember to aliquote the buffer into small volumes. Only have one aliquote as working will the rest remain frozen. Neither buffer nor enzyme likes freeze thaw cycles.
Now another thing to look at is the PCR insert, NheI needs a little more bp skirting the restriction site to work. I would give it at least 6bp if not 8bp. How many bp do have surrounding the NheI site
Thanks a lot for all your suggestions. Yes, I did not dephosphorylate the vector so I do not have issues of overtreatment with phosphatase. The NheI overhang has 3bp skirting the NheI site - I will repeat the cloning with a new primer that has atleast 6-8 bp. Is there a particular "skirt" sequence of the extra 3-5 bp that I have to use or can I just include any random sequence of nucleotides?
okay good luck. NEB technical reference is a good place to find out information on enzymes. Look up the cleavage to close ends link as well. NheI can barely cut with only 3bp skirting the restriction site.
As for the skirting bp, almost any base pair sequence will do. However,
1- make sure those bp do not introduce any sequence that cause self complementation of the primer, ie hairloops. I use Oligocalculator to check for primer self complementation (and primer melting temperature)
2- the ends shouldn't be AT rich. It should lean more towards GC, as that helps with the binding of the 5' end primer during the mid phase of the PCR.