helencollins560

Hi

just performed and RT and following real-time pcr using 10mM dntp instead of 2.5mM dNTP for both RT and realtime

just wondered whether i would get any usable data from that? or whether as my gut tells me that i will need to complete the plates again

any help, advice would be much appreciated

Dear All

I need to perform an endpoint PCR to test whether i have knocked out a specific gene in the heart.  My gene of interest has been floxed and subjects with this floxed gene have been crossed with cardiac specific MHC alpha cre mice and offspring genotyped.

I wondered whether i could use DNA extracted from cardiac tissue (qiagen gentra pure gene) with PCR primers for my gene of interest and MHC (in a similar manner to processing tail snips) or whether i would need to perform an RNA extraction then a reverse transcription to create cDNA then PCR.  I usually do alot of realtime PCR so my knowledge of endpoint PCR in comparison is lower and wanted an outside opinion.

I also have two sets of primers for my gene of interest- [1] genotyping ones which consist of WT and flox (and also a KO primer which i do not use) and [2] primers for my gene of interest that i designed for RT-PCR.  I assume if i have knocked my gene of interest out that both sets of primers would give me the same results.

I also thought that if i performed a real time PCR that i could infact obtain realtime data and then run the products on a gel also

any help, advice etc that anyone has will be appreciated

I need to make a 40% acrylamide stock (100g acrylamide/bisacryl (19:1) in 175ml distilled water)

however, we do not have any acrylamide/bisacryl (19:1) in powered form, but we have plenty of biorad 30% acrylamide/bisacryl (29:1) solution is there any way of me being able to make the solution i need using this? if so, how would i go about doing that?


i was thinking i could either purchase some acrylamide/bisacryl (19:1) in powered form or 40% acrylamide/bisacryl (19:1) liquid form


any advice would be appreciated!!!

I will be go vaildating my real time primers/probes before I  use them on "real" samples.
I have been told to run the product on a PAGE gel, however, is it also possible to use an agarose gel?

If not does anyone have a good recipe for a PAGE gel.

I have primers/ probes for mouse tissue, however, i wondered how to go about finding out whether these could also be compatible with rat tissue or if not, what software i could use to design some.

many thanks!