I am trying to do long distance inverse PCR (LDI-PCR), which requires restriction digestion of genomic DNA. I tried digesting my genomic DNA with BglII, XbaI and BamH1. I have the picture of the agarose gel attached to show you how it looks like after 2 hours digestion. gDNA1-4 are from the same cell type just different way of DNA isolation. Could anybody tell me if my digestion worked? Or what a good digestion pattern should look like? Thanks so much!
I designed an experiment to identify chromosomal translocation involving gene of interest.
Since the fusion gene contain my gene of interst (X) and an unknown partner gene, I plan to use a PCR+cloning+nested PCR strategy:
1)PCR using one primer hybridizing gene X and a combination of 3 short random primers. PCR is carried out at a low anealing temperature (42 degree)
2)The PCR products are then cloned into a T-A vector, transformed and individual clones are grown to get plasmid DNA. I expect a lot of clones are just non-specific, meaning that they do not even contain any part of gene X.
3)Do a second round PCR using an nested (inner) primer specific to gne X in combination with either T7 or SP6 promoter primer, which hybridize the T-A vector itself. Therefore,the clones do not contain gene X will not be amplified. And I expect that the PCR product should contain at least a portion of gene X.
4)PCR products from step 3 are cloned into T-A vector again and sequenced to identify fusion genes.
Does this stratagy sound OK? My currunt problem is that the majority of the clones from step 4)have nothing to do with my gene of interest. Any comments are greatly appreciated. Or drop me a message if you are interested in knowing more details. Thanks a lot!
I am trying to measure IgG secretion of cells in culture. I need to take samples every 2 days during an 8-day period. I am wondering if I could collect the samples and freez them untill the end of the culture and then measure them all at once? Thank you!