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removing part from plasmid


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18 replies to this topic

#1 lucilius

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Posted 26 May 2014 - 12:18 PM

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?

#2 bob1

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Posted 26 May 2014 - 01:23 PM

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.



#3 phage434

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Posted 26 May 2014 - 02:53 PM

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.



#4 neuropath

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Posted 26 May 2014 - 04:59 PM

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-...osts/14618.html

 

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

 

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#5 lucilius

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Posted 27 May 2014 - 01:33 AM

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-...osts/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.



#6 Trof

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Posted 27 May 2014 - 02:32 AM

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.

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#7 phage434

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Posted 27 May 2014 - 03:07 AM

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.



#8 Trof

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Posted 27 May 2014 - 03:48 AM

Yes, the phosphate thing I was wondering about.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#9 lucilius

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Posted 27 May 2014 - 03:49 AM

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?


Edited by lucilius, 27 May 2014 - 03:52 AM.


#10 bob1

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Posted 27 May 2014 - 12:49 PM

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 Files



#11 lucilius

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Posted 28 May 2014 - 03:00 AM

Yes, the phosphate thing I was wondering about.

 

Yes me too.

 

1) the plasmid itself will be phosphorylated at the 5' no? So I am guessing its because of the 3' part of my vector that has to ligate with the 5' part of the oligo?

2) do they have to be phosphorylated to anneal them? This is something I wonder about. Do strands of DNA need to be phosphorylated to anneal ??

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.

 

Ok thanks for the protocol, I'll check it.

 

PAGE? Not sure what that is, I'll check it. But I'll remind that they have to be purified a lot better than.

But you mentioned: they have to be 5' phosphorylated? Even if my vector is phosphorylated? Or is the vector not phosphorylated after I digest it with an RE? I guess it is because the 3' of my vector has to ligate with the 5' of the oligo and thats why?



#12 phage434

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Posted 28 May 2014 - 03:10 AM

You are corrrect that this might work without the phosphorylation of the oligos. Phosphorylation is not necessary for annealing, but is for complete double stranded ligation. With phosphates only on the vector, there will be nicks on the opposite strand. Since the overhangs are quite long (probably 20 bp) these nicks can be fixed by E. coli cells when transformation is done, but this will be less efficient.

Your major problem will likely be background from uncut or partially cut plasmid transforming. Be prepared for examining many colonies to find correct ones. This is the major advantage of the PCR approach I suggested earlier -- you can amplify very small amounts of vector, and then cut that template vector with DpnI, dramatically reducing background.

While PAGE purification would be nice, I don't think it is absolutely necessary.



#13 lucilius

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Posted 28 May 2014 - 08:09 AM

You are corrrect that this might work without the phosphorylation of the oligos. Phosphorylation is not necessary for annealing, but is for complete double stranded ligation. With phosphates only on the vector, there will be nicks on the opposite strand. Since the overhangs are quite long (probably 20 bp) these nicks can be fixed by E. coli cells when transformation is done, but this will be less efficient.

Your major problem will likely be background from uncut or partially cut plasmid transforming. Be prepared for examining many colonies to find correct ones. This is the major advantage of the PCR approach I suggested earlier -- you can amplify very small amounts of vector, and then cut that template vector with DpnI, dramatically reducing background.

While PAGE purification would be nice, I don't think it is absolutely necessary.

 

The transformation check is something I wonder about: is there an easy way to check for the correct ones? Or do I have to miniprep many of them and have them sequenced?


 

The PCR approach: I might try it, but I think its hopeless. Its a plasmid of about 12Kbs and it contains a lot of hard to amplify parts. If I use a PCR to generate the plasmid I will have too many mistakes I think.



#14 phage434

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Posted 29 May 2014 - 03:10 AM

For PCR: Use Q5 or Phusion in the GC rich buffer.  Add 5% of a 1 M betaine solution.

 

For detecting correct clones, a colony pcr works usually. One primer on the insert, another on the vector. Pick a small amount of a colony into 50 ul of water. Use the same tip to drop 5 ul onto a gridded petri dish. If you feel confident, you can also grow up a 2 ml culture in preparation for a miniprep run.

Add 1 ul of the water samples to a 10 ul PCR reaction. Cycle with a 5 minute initial 96-98 "boiling" cycle, followed by normal PCR.



#15 lucilius

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Posted 29 May 2014 - 11:49 AM

For PCR: Use Q5 or Phusion in the GC rich buffer.  Add 5% of a 1 M betaine solution.

 

For detecting correct clones, a colony pcr works usually. One primer on the insert, another on the vector. Pick a small amount of a colony into 50 ul of water. Use the same tip to drop 5 ul onto a gridded petri dish. If you feel confident, you can also grow up a 2 ml culture in preparation for a miniprep run.

Add 1 ul of the water samples to a 10 ul PCR reaction. Cycle with a 5 minute initial 96-98 "boiling" cycle, followed by normal PCR.

 

The PCR: I will keep it open as an option, but I doubt it will work since I really have a large and difficult plasmid.

I also wonder: I would first cut the part out (restriction reaction) , put it on gel and then do the PCR on the cut plasmid?

 

 

For the correct clones: you are talking about a vector in which you have added a part? My first idea was just to remove a part (fill it in with T4 polymerase), not really to add anything at first. I am guessing that only transformed cells with a closed plasmid would survive?

 

The second step would indeed be inserting a part, but would I not see this on a gel prior to transforming my cells?






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