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RE digestion and ligation troubleshooting.


Best Answer phage434, 23 April 2015 - 05:04 AM

At this point, the cost of doing an additional ligation is low. I would try all three of your AP enzymes in parallel. You need only about 20 ng of vector DNA in the ligation reaction, so you likely have enough. Don't overdo the AP treatment. Small amounts of enzyme for small amounts of time. CIP, in particular, has a bad reputation for trashing DNA, and can't be heat inactivated. SAP can be heat inactivated, which makes it much easier to use, since you can go directly from your digested DNA to the ligation without purification steps. If you use an enzyme that can be heat killed, then you don't need to purify the cut plasmid or the dephosphorylation reaction.

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#1 Wek

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Posted 22 April 2015 - 07:26 PM

I am trying to ligate my insert of 1kbp into a vector of 4-5kbp but I am having some trouble. Here are the steps that I follow:

 

For the insert:

 

Using NotI I digest my insert out of another plasmid, run the digested product on a gel and I see two bands where expected. I cut the band with my insert, gel purified (Qiagen) and put in -20C until needed.

 

For the plasmid:

 

Using NotI I digested the plasmid, run the digested product on a gel, an see only one fairly clean band where expected. There are no smears only a tiny "tail" on each edge of the band (probably due to too much DNA?). I cut the band, gel purified (Qiagen), and put in -20C.

 

The first time I did this I did not dephosphorylate my vector and none of the colonies I screened had the insert.
This time I couldn't dephosphorylate after gel purification due to time constraints so I threw it into -20C hoping it would not re-ligate if frozen. I plan to treat my vector with CIP tomorrow morning. Would I encounter problems during ligation because I waited 12-18hrs to dephosphorylate my vector?

 

During my first try, both my insert and vector after gel purification had a 260/280 of 1.8-1.9 but the 260/230 was <1.0 (I think mostly likely due to salts and not ethanol). I was told to ignore the 260/230 ratio and go ahead with the ligation. Could these salts cause problem? I know I end up diluting the salt with H2O and ligation buffer but I have no experience with cloning and this is becoming frustrating.

 

Also, would it be worth using CIP/AP/SAP that has expired? I think the SAP I found expired a decade ago, AP expired 4 years ago and there's no expiration date for CIP.


Edited by Wek, 22 April 2015 - 08:10 PM.


#2 phage434

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Posted 23 April 2015 - 05:04 AM   Best Answer

At this point, the cost of doing an additional ligation is low. I would try all three of your AP enzymes in parallel. You need only about 20 ng of vector DNA in the ligation reaction, so you likely have enough. Don't overdo the AP treatment. Small amounts of enzyme for small amounts of time. CIP, in particular, has a bad reputation for trashing DNA, and can't be heat inactivated. SAP can be heat inactivated, which makes it much easier to use, since you can go directly from your digested DNA to the ligation without purification steps. If you use an enzyme that can be heat killed, then you don't need to purify the cut plasmid or the dephosphorylation reaction.






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