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Subloning problems - 1.7kb and 10kb cloning problems (Jul/14/2009 )

Hello , Im having problems with a cloning experiment and lm getting to the suicidal point. heheheheh.

I have a 16kb vector which is linearized with SacII (cohesive end) and my insert is 1.7kb long, with SacII cohesive ends. Eventhough this could sound like an easy ligation is not working. I have tried everything.

Ive tried everything vector:insert ratio 1:1, 1:3, 1:5, insert:vector ratio 1:1, 1:3, 1:5. and I cant get positive clones.
First, I linearized my vector and tried to purify it with the Qiagen qiaquick colum system... doesnt work for Dna fragment that long, gives a poor yield, so im performing now electroelution, the yield is better. After the digestion, and purification, I perform a desphosphorilation with CIAP, 1hr 37C, then quantify the DNA and then the ligation with my 1.7Kb insert

Ive been using T4 DNA ligase from Fermentas that provides a 10X buffer and allows to use more DNA in a less total amount of reaction.

Id like to know if someone knows something about this, or long fragment ligation problems?
or if someone knows something about an special bacterial strain that can handle this experiment? or a Ligase that is better suitable for this?!
I would really appreciate the info
Greets

-soldierhxc-

Try this:

Digest the vector, CIP the digests and then precipitate everything. Resuspend in 30 ul water, estimate the conc and then setup the ligation with the purified insert. This should work, you would have religations but will surely get a clone as well. For large fragments, this works very well.

Best,
TC

soldierhxc on Jul 14 2009, 10:24 PM said:

Hello , Im having problems with a cloning experiment and lm getting to the suicidal point. heheheheh.

I have a 16kb vector which is linearized with SacII (cohesive end) and my insert is 1.7kb long, with SacII cohesive ends. Eventhough this could sound like an easy ligation is not working. I have tried everything.

Ive tried everything vector:insert ratio 1:1, 1:3, 1:5, insert:vector ratio 1:1, 1:3, 1:5. and I cant get positive clones.
First, I linearized my vector and tried to purify it with the Qiagen qiaquick colum system... doesnt work for Dna fragment that long, gives a poor yield, so im performing now electroelution, the yield is better. After the digestion, and purification, I perform a desphosphorilation with CIAP, 1hr 37C, then quantify the DNA and then the ligation with my 1.7Kb insert

Ive been using T4 DNA ligase from Fermentas that provides a 10X buffer and allows to use more DNA in a less total amount of reaction.

Id like to know if someone knows something about this, or long fragment ligation problems?
or if someone knows something about an special bacterial strain that can handle this experiment? or a Ligase that is better suitable for this?!
I would really appreciate the info
Greets

-T C-

digest, cip and then precipitate? without cleaning ?? no phenol:chloroform??

What conc ratio would you recomend for this vector 16kb insert 1.7kb???

-soldierhxc-

Hey,

When u precipitate, you would go for an ethanol wash anyways. No clean up required. I generally use more of the insert than the vector and try 2-3 combinations. Sorry can't tell u t eh exact conc. as I just run 3 ul of each on the gel and visually determine how much of the vector and insert to be used.

Best,
TC

soldierhxc on Jul 15 2009, 08:45 PM said:

digest, cip and then precipitate? without cleaning ?? no phenol:chloroform??

What conc ratio would you recomend for this vector 16kb insert 1.7kb???

-T C-