ligation question! - (Apr/14/2006 )
i use Creator SMART construct my cDNA library,when my cDNA ligate to digated de-phosphorylation vector and transform to E.coli DH10B,but when i pick the clonoy to PCR anylsis the insert and there is no insert. All the same to origial vector .So i don't konw where is the question.my vector and cDNA all digation with SfiI Enzyme which cut two places product the sticky detail.
-wang_gold-
QUOTE (wang_gold @ Apr 14 2006, 06:07 PM)
i use Creator SMART construct my cDNA library,when my cDNA ligate to digated de-phosphorylation vector and transform to E.coli DH10B,but when i pick the clonoy to PCR anylsis the insert and there is no insert. All the same to origial vector .So i don't konw where is the question.my vector and cDNA all digation with SfiI Enzyme which cut two places product the sticky detail.
Libraries and site-directed mutagenesis are the most difficult lab challenges;) Don't worry. Do you screen your clones using any selective media (eg. contain X-gal and IPTG). Or do you check by PCR all your clones?
-geneticcat-
QUOTE (geneticcat @ Apr 14 2006, 09:17 AM)
QUOTE (wang_gold @ Apr 14 2006, 06:07 PM)
i use Creator SMART construct my cDNA library,when my cDNA ligate to digated de-phosphorylation vector and transform to E.coli DH10B,but when i pick the clonoy to PCR anylsis the insert and there is no insert. All the same to origial vector .So i don't konw where is the question.my vector and cDNA all digation with SfiI Enzyme which cut two places product the sticky detail.
Libraries and site-directed mutagenesis are the most difficult lab challenges;) Don't worry. Do you screen your clones using any selective media (eg. contain X-gal and IPTG). Or do you check by PCR all your clones?
thanks to geneticcaat.
i use the chloramphenicol to screen my clones, i pick all the clones to PCR but there were no inserts. And now i do the Enyzme digation and ligation again.How do i decide the ligation proportion between the vector and inserts DNA?
-wang_gold-