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removing part from plasmid - (May/26/2014 )

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Hallo all, I have a plasmid that is good to use as a backbone for future cloning, however it contains a part I would like to remove. Now my plan was the following: - remove part I dont want with 2 restriction enzymes - fill in the sticky ends (but RE give sticky ends that dont "match") - religate the plasmid. My questions now were: whats the best strategy to fill in the sticky ends? I was thinking about mung bean nuclease but I often read its not the best option? Should I just just klenow polymerase? (it does not really matter for the reading frame how I blunt it). I also found something called "end-it dna end repair kit" but I have no experience with this. To religate the plasmid: is the 5' end still phosphorylated after I use mung bean nuclease/knelow or end-it dna end repair kit ? I was thinking to use T4 ligase to ligate the plasmid. Any insights?

-lucilius-

Most people would use the Klenow fragment of  T4 DNA polymerase to fill.

 

Another option is to ligate in a short adapter sequence - this gives you the option of adding in any restriction sites you might want to use.

-bob1-

Another (easier in my opinion) technique is to use PCR to ampllify the plasmid portion you want to keep. You can add whatever RE sites you need to the 5' ends of the priimers to allow you to make arbitrary cloning sites, or simply to let you religate easily after digestion.

-phage434-

Or you can just incubate it in a PCR reaction without any primers. Remember NOT to use Taq polymerase though as it adds an 'A' overhang at the end

http://www.protocol-online.org/biology-forums/posts/14618.html

 

You didn't mention if the overhangs created by your restriction digest are 3' or 5'.

 

~neuropath~

-neuropath-

Most people would use the Klenow fragment of  T4 DNA polymerase to fill.

 

Another option is to ligate in a short adapter sequence - this gives you the option of adding in any restriction sites you might want to use.

I guess the T4 Polymerase is the best option than!

It leaves the phosphate groups at the 5' I hope? So I can simple ligate with T4 ligase afterwards?

 

Another (easier in my opinion) technique is to use PCR to ampllify the plasmid portion you want to keep. You can add whatever RE sites you need to the 5' ends of the priimers to allow you to make arbitrary cloning sites, or simply to let you religate easily after digestion.

 

Sadly a PCR is out of the order here because the plasmid is pretty large and it contains some regions that are very GC rich and or T rich. I am having problems just getting it sequenced so a PCR strategy wont be very good to use.

I also dont need to put something in the place where I remove the part. That part is really just a part I need to cut out, but thats just it, I dont need that place for further research.

Or you can just incubate it in a PCR reaction without any primers. Remember NOT to use Taq polymerase though as it adds an 'A' overhang at the end

http://www.protocol-online.org/biology-forums/posts/14618.html

 

You didn't mention if the overhangs created by your restriction digest are 3' or 5'.

 

~neuropath~

Perhaps this is an alternative for the T4 ligase, I'll keep it in mind.

-lucilius-

I just wondered if making a short linker cassette would be also possible.
 
like if you have this in digested plasmid:

AGT-3'        5'-ATGCAT
TCAGCAT-5'      3'-CGTA
Buing two oligos that would make this:
CGTA..........   
    ..........TA           
You can add anything in the middle for example new restriction site, as long as both oligos are around 20-30, which is the comon length for primer design.

-Trof-

Don't forget to order those adapter oligos with 5' phosphates, or use PNK to add the phosphates. Remember when you do the ligation that you will need seemingly vanishingly small amounts of your annealed oligos (near equimolar, which means very low weight). And do anneal them prior to trying the ligation.

-phage434-

Yes, the phosphate thing I was wondering about.

-Trof-

I just wondered if making a short linker cassette would be also possible.
 
like if you have this in digested plasmid:

AGT-3'        5'-ATGCAT
TCAGCAT-5'      3'-CGTA
Buing two oligos that would make this:
CGTA..........   
    ..........TA           
You can add anything in the middle for example new restriction site, as long as both oligos are around 20-30, which is the comon length for primer design.

 

 

I am not very familiar with this. I know about it, but how does it work from a practical standpoint?

You order two oligos and then? How do you make them ligate to eachother?

Don't forget to order those adapter oligos with 5' phosphates, or use PNK to add the phosphates. Remember when you do the ligation that you will need seemingly vanishingly small amounts of your annealed oligos (near equimolar, which means very low weight). And do anneal them prior to trying the ligation.

So I would need very low amounts of oligos to anneal into my cut plasmid?

 

 

I could do it like this, but not sure its worth it because I really dont need that site of the plasmid for further research, but it might be worth it.

 

BTW: is it necessary to have them phosphorylated ? if I do not dephosphorylate my vector, than why should I?

-lucilius-

Annealing any oligos is very simple and quick - it is what I was talking about in my post above.     All you have to do is put them in a buffer, heat to 95 for a couple of minutes in a heat block and then allow to cool to room temperature slowly.  I've attached a proper protocol.

 

The trick is  that the oligos must be PAGE or better purified and be 5' phosphorylated so that the ligations will work - the phosphorylations are needed so that the ligation process can proceed - the phosphates are hydrolysed by the ligase to provide energy for the ligation reaction (IIRC).

 

If your vector doesn't have complementary ends, there is no need to dephosphorylate.  Phosphatases often are hard to deactivate and cause problems in downstream reactions.


Attached File

-bob1-
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