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Difficulty cloning vector with multiple Lox sites


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#1 AVG77

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Posted 03 September 2009 - 06:45 AM

HI,
I am trying to clone a big chunk of insert (5kb) with multiple lox sites in a 10Kb vector. Both the insert and vector is RE digested and blunted using NEB's quick blunting kit. Followed by that vector has been dephosphorylated using NEB Antartic Phosphatase. Finally the product is clean and single band on the gel. A o/n ligation was set up and transformed next day using DH5a CC, plated on Amp plates.
I got more (2X) colonies on self ligation than vector plus insert.
The original vector from which I cut open the lox cassette has a very poor growth in bacteria,
Does anyone has experienced cloning problems like this or has any suggestions for me to handle this cloning differently than what I am doing right now...
I badly need this clone and have been working on this for last six months,
Any suggestions will be of great help,

#2 Warren

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Posted 05 September 2009 - 11:22 AM

HI,
I am trying to clone a big chunk of insert (5kb) with multiple lox sites in a 10Kb vector. Both the insert and vector is RE digested and blunted using NEB's quick blunting kit. Followed by that vector has been dephosphorylated using NEB Antartic Phosphatase. Finally the product is clean and single band on the gel. A o/n ligation was set up and transformed next day using DH5a CC, plated on Amp plates.
I got more (2X) colonies on self ligation than vector plus insert.
The original vector from which I cut open the lox cassette has a very poor growth in bacteria,
Does anyone has experienced cloning problems like this or has any suggestions for me to handle this cloning differently than what I am doing right now...
I badly need this clone and have been working on this for last six months,
Any suggestions will be of great help,


You may want to give some more details as to your process ("the product is clean" does that mean you did some purification?). However some things you can try -- use a different cell type and/or lower temps (ie 30) for this construct. IF you can I would use restriction sites instead of blunt cloning -- this is hard enough with small inserts! I would use low temps for the ligation plus PEG in the ligation buffer, both of which will help blunt end ligation. You might need to purify your vector and insert by some method after the klenow blunting, because the dNTPs may interfere with the ligase. The dNTPs are also substrates for the phosphatase, which may be why you do not see complete dephosphorylation of your vector. Gel purifying such large fragments is not ideal, so i would use a different method. Finally, I would just set up a series of smaller ligation reactions (instead of one big one) and vary the amount of template and vector over a wide range. For some reason this was the trick when I tried to do something similar years ago, and when it worked it worked well (ratios were correct), when it didn't work well, I got nothing!

Warren..

#3 eldon

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Posted 06 September 2009 - 07:55 AM

HI,
I am trying to clone a big chunk of insert (5kb) with multiple lox sites in a 10Kb vector. Both the insert and vector is RE digested and blunted using NEB's quick blunting kit. Followed by that vector has been dephosphorylated using NEB Antartic Phosphatase. Finally the product is clean and single band on the gel. A o/n ligation was set up and transformed next day using DH5a CC, plated on Amp plates.
I got more (2X) colonies on self ligation than vector plus insert.
The original vector from which I cut open the lox cassette has a very poor growth in bacteria,
Does anyone has experienced cloning problems like this or has any suggestions for me to handle this cloning differently than what I am doing right now...
I badly need this clone and have been working on this for last six months,
Any suggestions will be of great help,


when you cut your vector are you using a RE that produces a blunt cut (e.g. SmaI)?

how are you transforming your cells? heat shock or electroporation? since vector size affects TE's, a self-ligated 10kb vector will transform better than a 15kb vector with insert. i recommend electroporation when your vector gets this large.

if the cells don't like the vector do you know why this might be? try SURE cells or maybe Stbl2 or Stbl3.

if the cells don't like the vector due to regions of low complexity etc., you can lower the growth temp to room temp...it takes longer for colonies to appear, but it can make a vector more tolerable to the cells. you can also reduce the AMP in the plates by half which also helps in propagating vectors the cells do not like. i have cloned 10kb cDNAs successfully into 10kb vectors by adjusting these parameters when using "normal" conditions failed (e.g., AMP concentration and culturing at 37C).




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