I've been trying to clone my N-terminal inserts in the comercial pEGFP-N1 vector.
Initially, I cloned my N-terminal insert into a pGEMT-easy vector to ensure that the insert digestion was done correctly. The enzymes I use for digestion are NheI and BamHI. Then, the inserts are purified using an agarose gel and an agarose gel extraction kit.
The vector is digested with the same enzymes and also purified in the same way. For some reason, when quantifying the DNA, the 260/280 ratio gives very high values (reaching values of 6 or 14). Then, a 1:3 ligation of vector:insert is performed and bacteria are transformed. So far, there have been no positive clones.
Im really worried cause I've been stuck for a really long time with this cloning, and I dont know what to now.
I think the high 260/280 ratio is telling you that there is something contaminating your DNA, possibly a reagent from the agarose gel purification. I think it best to run a small amount of your purified fragment on a gel to be sure you have intact DNA. Whatever this is might also be inhibiting the ligation since it is likely organic chemical.
However you can also try the cloning without gel purification. Since pGEM-T is Amp resistant and pEGFP-N1 is (I think) Kan resistant, you can select solely for the recipient vector alone. I frequently just digest both DNAs with the enzymes, run an aliquot on a gel to confirm they are well digested then set-up your ligation. Now the trick is how to you remove the background of vector that does not have the insert. There are many restriction sites in the polylinker that will be present in the intact plasmid but will now be deleted in the recombinant (just make sure they are not on your insert) such as PstI or EcoRI and then digest your ligated product with this enzyme to remove the background of vector alone. This is an approach that has often worked well for me.
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