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pcr on genomic dna ( tailsmouse), HELP !!!!
2 replies to this topic
Posted 13 June 2001 - 09:00 PM
Hello,I'm doing some pcr on genomic dna from tails to screen some positives transgenic mice but much problemes about :1)genomic dna extraction protocol: much protocols on litterature without and with phenol/chloroform2)About PCR: all my controls are OK but one day I have a positifand a day later it's negatif !!!The samples are stored at +4°CHelp! dear molecular biologist !!! Thanks
Posted 18 June 2001 - 09:00 PM
I digest 2mm tail snips with a solution of ProK and PBND. Digest overnight. I use 12ul reactions using 2ul of DNA from the digestion. The problem sometimes is that you might have too much DNA from tails and it will tend to give you results one day and no results the next day, like what you are having. You need to get consistent yeilds in order to get consistent pcr products.