Dear all !
I have hard time in ligation . i cut PKH3 vector and avian Rsk1 with single as well as double cut simultaneously with BamH1 and Hpa1 .when checked bands were expected.but when i used to ligate vectorPKH3(4.2kb) with insert RSK1(2.8kb) and PKH3(4.2KB) with PKH3( 600bp) together as +ve control but i couldnot get any colony after transformation .my competent cell and ampicilline plate has no problem as i checked with another plasmid DNA. i took 1:3 vector and insert ratio.still i didnot succeed in this experiment. i did as you suggested me previously .but unfortunately no colony appeared. therefore , plz suggest me again so that i can overcome my problem.
By the way, how can i find molar ratio of vector PKH3(4.2KBP) and avian RSK1( 2.8KBP) ? i found vector concentration 220ng/microliter and 75 ng/microliter of insert RSK1 after measuring.
how can i know whther my ligase and ligase bfr isworking or not , is there any method to check or we we have to check with new bfr and ligase?
Iam waiting your kind cooperation.
I hope you will writr me very very soon .
Thanking you again
ligation and transformation
Dear all !