sligeach

I am also currently cryosectioning some skeletal muscle sections and i am gettting some horrendous results with lots of holes in my samples presumably from ice crystal damage. The samples i am currently working on i did not freeze down myself however i asked the people who did freeze them down and the protocol they followed seems pretty standard ie freeze samples in liquid nitrogen cooled isopentane and then store at -80 till use. they did also cover the samples in oct before freezing down so maybe this has a negative effect. Is there a certain time frame the sample should be left in isopentane for?

However one thing that i would be interested to hear peoples thoughts on is wold the cryostat have any effect on the formation of the many muscle tears i see in my muscle sections persumably caused by ice crystal formation. I have recently moved to a new lab and the cryostat that we have here is one i dont like its a small box shaped one which can fit onto a table top and so i worry about how good its temperature regulation is as when you take off the lid the sample is quite close to the "outside" ie room temp. In my previous lab we had a bigger cyrostat with a sliding door and the sample was mounted a larger distance from the "outside" of the cryostat.

A second thing i was wondering about is the air drying of the sample. Once i cut my tissue section and have it on my slide can i leave that slide then straight away at room temperature for the sample to air dry and how long should i do this for? I have typically been leaving the sample for 10-15mins at room temp post cutting and then fixing in 2% paraformaldehyde.

hi battery horsestaple

I am currently genotyping some transgenic mice for the presence of a floxed allele and i have observed something almost identical indeed i have just today posted a question similar to yours on this forum. In my case i have now used three different primer sets which flank the floxed insertion allele and i often get a third allele which is 300bp longer than wild type but about 1000bp shorter than the floxed allele. I had thought something weird must have happened during recombination but i cant get a satisfactory answer so hopefully somebody may be albe to find an answer as it would be very much appreciated. I am intending to cut out a third allele band and get it sequenced just to see what exactly it is as i hate not knowing.

thanks for all the suggestions. I thought i would just let people know who may be having similar problems to what i had - that i decreased the DNA concentration to 10ng per 25ul total volumer pcr reaction and most of my PCR reactions worked reasonably well. there are still a few samples that are not working very well so am still trying to optimise these.

yes from my work with RNA and qPCR i learned very early the importance of always running temp gradients to optimise the primer sets and i also test out different primer concentrations of 0.5 1 and 2um as from my experience some primer sets need a higher concentration in order to give me consistent and satisfactory detection at least for lowly expressed mRNA transcripts.

For genotyping experiments im beggining to think it may be worth the extra expense of buying a commerical DNA extraction kit in order to ensure or at least improve the likelihood of me getting consistently useful DNA for PCR experiments as the amount of time i have spent trying to get the current DNA samples working is not cost effective as ive had to use signficantly more of my time and PCR kit than i had planned.

If anybody has experience of using a commercial DNA extraction kit and found it to consistently give them good quality/pure DNA from mouse tail extractions for subsequent PCR then i would be very interested to hear about it.

thanks for your reply bob1

Yes my negative controls are negative

I have tried multiple different primer sets 4-5 and i keep getting the same pattern in that for the samples which give a good signal for my positive control primers i get expected bands with the other sets of primers while for the samples which give low or no signal with the positive control primers i typically dont get any signal with the other primer sets. It was the latter finding which made me realise it must have something to do with the actual DNA template and this was confirmed with the positive control primers.

i was thinking of doing a dilution and seeing what would happen.

For your genotyping experiments bob1 do you have a preference for a particular pcr kit or DNA extraction method and also what concentration of DNA and primers do you typcially use. I had a 25ul pcr reaction with 250ng of DNA and 0.5um primers.