Hello Researchers,
I am trying to clone my target genes into a promoter region of a vector (pcDNA3.1-GFP). I use 1 ug/ml of vector for restriction (20 ul volume) and I follow Invitrogen purelink gel elution kit to elute the restricted DNA. Whenever I try to elute in the gel without EtBr (0.8%), I not only fail to get high yields of restricted DNA but also observe a smeared band indicating the possibilities of DNA degradation. I therefore take steps to get rid of DNAse contamination. Any suggestions or tips are welcomed to troubleshoot this and thanks in advance.
Hi Snehaa,
It would be great to see the picture, and I am not sure, that you have a degragation. Restricted vector itself may form additional bands in gel.
I'm not familiar with that exact kit, but do you use DNase/RNase free Protinase K to degrade the nucleases?
When you stated you try to elute the nucleic acid (NA) from the gel without EtBr, are you sure your gel piece is the one that contains your NA? As Andrey mentioned, we need to see a gel pic to find out whether or not you actually got the DNA you needed. Be sure to check the gel without EtBr and the spot you excised the agarose from with a gel that shows where the band should be.
Hello everyone,
Thank you for your suggestions. The kit we use is fine and apt for the vector. Further we use sterile water for elution. More over, the kit works fine with other DNAs with same range of sizes. And, I have added a picture of the gel which may help yet more.
- Badges
- Science method