Ligation failed

newsnet customer customer at newsnet.com
Fri May 5 06:12:28 EST 2006


Hi,

I have transformed an insert/gene (2kb) into a cloning vector.
Then cut the gene out to ensure it has sticky ends.
Also, I have linearised an expression vector (11kb) with compatible sticky ends to the insert.

Here is the ligation reaction:

insert (12ng/ul)                     2ul
linearised vector (6ng/ul)        4ul
10X lig. buffer                       1ul
T4 ligase                              1ul
Water                                  2ul
TOTAL                                 10ul

overnight incubation @ 4C in accordance to Roche T4 ligase information sheet.
Total DNA in ligation reaction is 48ng.
insert:vector ratio is 1:1 in accordance to Roche T4 ligase information sheet when insert to vector size are significantly different.

ligation reaction was transformed into chemically competent cells Invitrogen One Shot TOP10 as according to protocol.
Incubation overnight showed no colonies.

HERE ARE MY THOUGHTS:
1. chemically competent cells are not suitable for vector >10kb - HIGHLY PROBABLE
2. ligase and/or ATP (in lig. buffer) is not functional/depleted - HIGHLY PROBABLE
3. Total DNA concentration is too high to promote circularization of vector - LOWLY PROBABLE AS STILL EXPECT SOME COLONIES

Interested to hear your thoughts.

Cheers,
ST






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