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you can start with 1% and go from there (when dialyzing you use equal concentration, the sds will be diluted by the dialysis solution).
are you sure your urea is intact. many people warm up their solution to completely dissolve urea quickly but that will actually cause the urea to decompose. the solution should be made at temperatures no higher than room temperature. it will take longer but it will be urea.
#107395 problem of 2D-GE with phagosome fractions
Posted
on 20 April 2011 - 08:24 AM
#107059 LIgation of Single stranded cDNA at both 5' and 3' ends with single str
Posted
on 17 April 2011 - 09:55 AM
Hello everyone,
I have prepared single stranded cDNA (ss cDNA) using random hexamers.
As expected the ss cDNA donot have 5'-p group.
I donot have any gene specific primers, as the cDNA was prepared after the RNA obtained from RNA immunoprecipitation (RIP).
I have to amplify the cDNA i have obtained from RIP. I hav a 3'-modified primer and a normal synthetic primer without any modification.
I have to ligate these primers to the two ends, bot 5' and 3'ends, of ss cDNA and use primers complementary to them for the amplification of ss cDNA by PCR.
can anyone please suggest how feasible is the process of ligation with T4 RNA ligase. And also please suggest whether to use both of them at a time during ligation or do a sequential ligation process. I have already 5'phosphorylated the modifed primer and also the ss cDNA, which is required during ligation.
please give suggestions and comments if any.
I have prepared single stranded cDNA (ss cDNA) using random hexamers.
As expected the ss cDNA donot have 5'-p group.
I donot have any gene specific primers, as the cDNA was prepared after the RNA obtained from RNA immunoprecipitation (RIP).
I have to amplify the cDNA i have obtained from RIP. I hav a 3'-modified primer and a normal synthetic primer without any modification.
I have to ligate these primers to the two ends, bot 5' and 3'ends, of ss cDNA and use primers complementary to them for the amplification of ss cDNA by PCR.
can anyone please suggest how feasible is the process of ligation with T4 RNA ligase. And also please suggest whether to use both of them at a time during ligation or do a sequential ligation process. I have already 5'phosphorylated the modifed primer and also the ss cDNA, which is required during ligation.
please give suggestions and comments if any.
