I am trying to insert shRNAs into lentiviral vector PLB (a modified plasmid of Pll3.7 and cloning protocol the same). I digested the plasmid with HpaI and XhoI. Those sites are immediately flanking the shRNA. The annealed DNA oligo has one blunt end (which is not HpaI site thus destroys the restriction site after ligation), and one XhoI overhang.
I did not dephosphorylate my vector and did not order my oligos as phosphorylated (because it's cheaper...). Thus it is possible that two or several vectors ligate together. I tried to confirm the colonies I got by doing the restriction digestion with HpaI and XhoI as the cloning protocol of Pll3.7 recommended. The protocol states: "Insertion of insert causes a band shift of ~60bp (100bp in my case) in an XbaI/NotI fragment when compared to parental vector. This can be seen by 2% agarose gel electrophoresis." Yet when I ran the gel (see attached picture), the digestion did not seem to work (left to right: cut PLB, cut ligated vector, uncut PLB, uncut ligated vector). I did the digestion for 1h, 2h, and 4h and the results are the same.
I am wondering what could cause this: the uncut vector does not seem to be bigger than uncut PLB, thus it is probably not two or several PLB ligated together. Even if it is the case, shouldn't the vector be cut to the same size as the linearized PLB?
Another possibility is that since the restriction site immediately follows the shRNA, could it be that the secondary structure of the insert causes the inefficiency of digestion?
It is also weird to me that the protocol says a 60bp or 100bp difference (out of 9kb) could be visualized on a 2% gel. When I ran a 2% gel the vector hardly migrates out of the well in 2h, 115V.
I extracted all the plasmids with Qiagen miniprep kit with an extra wash and eluted with EB.
I am going to get primers to confirm by PCR. But I still want to know what is going on with my digestion.
Advise and suggestions much appreciated!
P.S. Attached is my gel pic.
HpaI - 5'-..GTT^AAC..3' - gives a blunt end. The annealed DNA oligo has one blunt end but not to restore the HpaI. it not easy for you to tell a 60 bp or 100 bp shifting up from a big size vector. I suggest that the best way is to sequence the cloning sites to see whether the annealed DNA oligo is present but without any mutation introduced during this process.
İt's hard to say something that we dont know what you did step by step but i suggest you to Look an enzyme upwards and downwards of your inserT not with the enzymes that the inserT Have on it's Edges. you should have the vector sequence and map. look for multiple cloning site and choose a suitable enzyme and digest with it then make an agarose gel. with this way you Will control if you inserT your gene of interest. If you see a band the problem is with your enzymes that your insert has on it's Edges . May be a mutation occured and the enzyme sequence changed and that is the answer why you can not cut it. Try this and make a sequence analysis so you can see what happened clearly. Good luck
Sebastian, thanks for reminding me. I should have digested with Xbal and NotI instead of HpaI and XhoI. That was a stupid mistake ;P
S. Aktas: thanks you are right! I should have done a different pair of digestion.
Hi, Xiwei. I am wondering how you finally figured out this problem. Recently I am using pLL3.7 vector. Could you give me some suggestion when I use these two enzymes together (HpaI and XhoI). I am trying to digest the vector with these enzymes in a sequential digestion now. Do you have any suggestion? Thanks in advance! Xiwei Shan
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