ligation to transformation - (Oct/27/2005 )
Hi,
Past one and half month am facing problem in ligation to transformation in cloning host.
Two controls which were misguiding right from beginning are
1) getting surplus tranformants with supercoiled DNA
2) an uprise above the size of vector on agarose gel after ligation with insert in ratios from 1:1 to 10:1(Insert to Vector)
Concluded insert might be toxic or instable i did one experiment by taking vector alone cut with single digestion and ligation.
No colonies at all that too not even background colonies. Ligase which am using is fermentas new.
Kindly give me some suggestions.
Regards,
Madhy
-Madhy-
QUOTE (Madhy @ Oct 27 2005, 03:04 AM)
Hi,
Past one and half month am facing problem in ligation to transformation in cloning host.
Two controls which were misguiding right from beginning are
1) getting surplus tranformants with supercoiled DNA
2) an uprise above the size of vector on agarose gel after ligation with insert in ratios from 1:1 to 10:1(Insert to Vector)
Concluded insert might be toxic or instable i did one experiment by taking vector alone cut with single digestion and ligation.
No colonies at all that too not even background colonies. Ligase which am using is fermentas new.
Kindly give me some suggestions.
Regards,
Madhy
Past one and half month am facing problem in ligation to transformation in cloning host.
Two controls which were misguiding right from beginning are
1) getting surplus tranformants with supercoiled DNA
2) an uprise above the size of vector on agarose gel after ligation with insert in ratios from 1:1 to 10:1(Insert to Vector)
Concluded insert might be toxic or instable i did one experiment by taking vector alone cut with single digestion and ligation.
No colonies at all that too not even background colonies. Ligase which am using is fermentas new.
Kindly give me some suggestions.
Regards,
Madhy
Hi MAdhy,
Well, after reading your woe I thought I'd been there. So this is what I believe and I follow..
First for Ligation, You need:
FRESH - Dephosphorylated & gel extracted & purified Vector, gel extracted & purified insert.
(I'm using a ligase which is about 14 months old.)
Optimum temperature (O/N @ 12 - 14 Celsius works for me)
For Transformation, You need:
Optimum Ab concentration; Fresh SOC medium; TOPO cells (any competent cells stored @ -80 Celius all the time) and proper positive control.
I hope this helps
Viku
-Viku-