I use restriction enzyme cloning method, and I have been using new reagents e.g., competent cells, and ligase reagent because initially I thought the problem is that these reagents were outdated. Now with these reagents I attempted to clone my shRNA into the vector with 1:3 DNA:insert ratio, but I didn't even see a single colony.
First, what restriction enzymes were used to cut the plasmid DNA?
After confirming on an agarose gel that the DNA was digested as intended, how was the band purified once it was cut and extracted?
Secondly, how did you prepare the shRNA inserts? Did you anneal the oligo DNAs? Or another method?
Third, what reagents did you actually use for ligation? You are correct, the older ligation reagents may not work because they are depleted of ATP.
Fourth, what E.coli strain did you use? shRNA plasmid cloning often causes recombination problems with DH5alpha.
Hi Yohei,
I use EcoRI and AgeI enzymes to cut the plasmid DNA. After running on the agarose gel, I cut the 7kb band and purify it with gel extraction kit and quantify with Nanodrop.
And I anneal and phosphorylate my oligos, maybe now im thinking I will just anneal them and not phosphorylate them. And i use
And I use instant sticky ligase reagent which is new, and I use neb-5aplha competent cells (maybe thats the problem)? I can use other neb-10 beta or any other competent cells you recommend?
OK, Mavra. There are few solutions I can suggest. The plasmid you are using sounds like a pLKO.1 plasmid vector as inferred from the restriction enzyme sites. I use it often too.
If you are using a variant of the pLKO.1 plasmid without 1.8kb stuffer, I would recommend digesting with AgeI first and adding an additional EcoRI later rather than double digesting because AgeI and EcoRI sites are very close. And if it does not contain the 1.8kb spacer, it is difficult to determine if it has been digested by electrophoresis on an agarose gel, but these problems do not occur when the 1.8kb stuffer is existed.
And for E. coli, we recommend NEB Stable, which proliferates worse than NEB-5alpha but reduces unexpected recombination.
However, these are unlikely to be the cause of the absence of E. coli colonies.
My biggest concern is that the oligo DNA as an insert may not be annealed correctly. Could you tell me what buffer you used for annealing and what protocol you used?
You were concerned about the absence of phosphorylation, but since phosphate is present at restriction enzyme-digested ends, phosphorylation is not needed for the insert. Phosphorylation of the insert is necessary only if the backbone vector is phosphatase-treated.
Hello and thanks a lot for your prompt reply. Now I would not phosphorylate the oligos and just anneal them. I used to use this as my protocol for phosphorylation and annealing (protocol attached). Btw whats the best annealing steps you recommend? And yes I use plko.1 plasmid, I would also try to cut it first hy AgeI and then by EcoRI.
Phosphorylation of the oligos:
5ul Oligo 1, Forward (100uM)
5ul Oligo 2, Reverse (100uM)
5ul 10X T4 Ligation buffer (with 10mM ATP)
35ul ddH20
1ul T4 PNK Buffer (T4 polynucleotide kinase buffer)
And just to mention, I add nuclease-free water to my oligo's when I first received them to make the concentration of 100uM. And from there I phosphorylate them as mentioned above, and put in the thermomixer for 37C for 30 mins, 95C for 5 mins, and leave them at room temperature for 1 hour.
I'm not sure whether the T4 buffers are suitable for annealing DNAs. It seems to me that there is no problem, but it is still reliable to use the NEBuffer 2 supplied with the restriction enzymes, as the step of phosphorylation of oligo DNAs is not necessary.
Furthermore, I do not recommend leaving the DNA at RT to be annealed, as it depends on the season and environment.
I have answered my protocol in a previous question, see below.
http://www.researchgate.net/post/How_can_I_clone_shRNA_for_gene_knock-down_in_pLKO1_vector
Perfect, I will do these steps in the coming week :) Thank you so much for all your recommendation and advice.
Lastly, if I don't have NEBuffer 2 can I mix both the oligos's, anneal them with the temperatures as you described and add water to resuspend them. Would they bind to each other without the buffer as they are complementary to each other?
I've never tried to anneal oligos without salts. You can easily make appropriate buffer in-house by just searching for "annealing buffer". Good luck!
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