# large fragment cloning problem! Help needed - (Jan/17/2008 )

I am doing the insert about 3.5kb(viral membrane genes) into a large vector(with viral genes) of 16.5kb with enzyme SacII and BsiWI. The insert which got from PCR products or double cutting from clone vectors were tried, the insert/vector ratio from 5:1~9:1, ligated with NEB T4 ligase for more than 24hours, afterthen, transformed into XL10-gold compentent cells. but unfortunately, positive colnes were not found. there were about 50-100 clones in the plate with Amp.
Even PCR positive were found, but the plasmid taken were so smaller than the vector.
Anyone can give me some suggestion?
thanks.
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-xfniu-

For the sake of illustration, I am going to assume that you used 5 ng of the 2500 bp insert and 1 ng of the 16500 bp vector (for a 5:1 ratio), or 9 ng of the 2500 bp insert and 1 ng of the 16500 bp vector (for a 9:1 ratio).

5 ng of the 2500 bp insert = 1.85 e 9 molecules (1.85 e 9 Sac I ends and 1.85 e 9 BsiWI ends)
1 ng of the 16500 bp vector = 5.6 e 7 molecules
9 ng of the 2500 bp insert = 3.34 e 9 molecules

Your 5:1 ratio is actually 33:1 and the 9:1 ratio is actually 60:1 for the number of ends you are trying to ligate.

Did you dephosphorylate your vector? Although double-cut, you probably have some single-cut vectors in the reaction and these can religate if you don't dephosphorylate (preferably with SAP).

When you check for inserts with PCR, you should use one primer from the insert and one primer from the vector.

-tfitzwater-

The ratio, I mean that the volume ratio for 3500bp insert(50ng/ul) and 16.5kb vector(50ng/ul) in total 10ul ligation mix, eg. 7ul:1ul.
There was clones find when vector only ligation transformed. But less than the insert-vector ligation.

Abosultely, insert and vector matched primers were used for PCR detection.

For more than 200 clones were detected, but no positive clones, even once, there was smaller one about 7kb missed plsmid were found with insert but some gene were missing.

QUOTE (tfitzwater @ Jan 18 2008, 07:13 AM)
For the sake of illustration, I am going to assume that you used 5 ng of the 2500 bp insert and 1 ng of the 16500 bp vector (for a 5:1 ratio), or 9 ng of the 2500 bp insert and 1 ng of the 16500 bp vector (for a 9:1 ratio).

5 ng of the 2500 bp insert = 1.85 e 9 molecules (1.85 e 9 Sac I ends and 1.85 e 9 BsiWI ends)
1 ng of the 16500 bp vector = 5.6 e 7 molecules
9 ng of the 2500 bp insert = 3.34 e 9 molecules

Your 5:1 ratio is actually 33:1 and the 9:1 ratio is actually 60:1 for the number of ends you are trying to ligate.

Did you dephosphorylate your vector? Although double-cut, you probably have some single-cut vectors in the reaction and these can religate if you don't dephosphorylate (preferably with SAP).

When you check for inserts with PCR, you should use one primer from the insert and one primer from the vector.

-xfniu-