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High background issue


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#1 plasmamembrane

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Posted 08 February 2014 - 03:46 PM

Hello,

 

    I am trying to prepare the vector for future cloning but bothered by high background. Below is my experiment setup.

 

   The vector is pFBDM, around 5.7Kb in size. I choose two restriction sites which span 45bp apart. One site is for XmaI , the other is for KpnI.

 

   I purchase two enzymes from NEB as fresh stock.

 

   I use 1ug of plasmid ( purified by qiagen mini-spin kit ) as starting material. Double digestion was setup at 37 degC, the solution volume is 50uL. 1uL (20U) of restriction enzymes were used in the digestion reaction. I digest the plasmid for 2 hrs. After the digestion, the mix was purified with Qiagen PCR clean up kit under the guidance from manufacturer that it can remove DNA under 100bp. The choice was under the consideration that UV exposure can markedly decrease the transformation efficiency and we don't have long-wavelength UV box in our lab. Another consideration is we plan to clone a fragment of 3.8Kb in size, so the efficiency is of upmost importance to us. After digestion, I test with gel; it looks good.

 

    I tested a aliquot of mixture for transformation. Surprisingly, I got hundreds of colonies ( DH5-alpha, efficiency around 5x10^7- 10^8 CFU/ug plasmid ). They are all uncut plasmids.

 

    I think the problems are 1. incomplete digestion of the plasmid 2. the kit did not remove the digested small fragments efficiently, so self-ligation occurs.

 

   Any suggestions for that? How to avoid gel-extraction? I know gel extraction might solve this issue, but I encountered marked decrease in efficiency. I had tested that.

 

Thanks a lot for your opinions.  

 



#2 phage434

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Posted 08 February 2014 - 05:48 PM

This is not too surprising. Cutting is never fully effective at removing background. I doubt if the presence of the short fragment makes much difference, since you have not ligated. Almost certainly the difficulty is uncut plasmid. In my experience, the best way of dealing with this is use PCR to amplify the plasmid. With PCR, you create double stranded, linear DNA fragments, which will not transform. Only when your insert is put together with the linear fragment and ligated, will you get a circular fragment which transforms. The PCR primers can be designed to have whatever restriction sites you want (you need 4-6 bases 5' of the cut site to allow restriction digest binding).

 

When you do the reaction, use very small amounts of the template (which will be circular and can transform). Following the PCR, purify the reaction (important!) and then cut with your enzymes and with the enzyme DpnI, which will cut the template DNA, but not your PCR product. If you have chosen enzymes which can be heat killed, then do so, and you need not purify, but can go directly to ligation.

 

It is sometimes possible to cut the linear PCR product and your insert in the same reaction by mixing (equimolar amounts) and cutting (if the insert has been made with a PCR reaction).



#3 plasmamembrane

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Posted 08 February 2014 - 06:46 PM

Thanks for your opinion. But amplifying 5.7Kb vector is a pain for me. Is their any other choices?

Perhaps I should gel purify the cut plasmid , however, the transformation efficiency is dramatically low after UV exposure.

 

I searched on internet and found some authors recommend the use of crystal violet or methylene blue to stain the gel thus avoiding the

UV exposure. Anyone has the experience on that?

 

Here is what I think. If I can visualize the bands under the visible light avoiding the UV, then I may get the cut vector.

Besides, is the Qiagen PCR purification kit compatible with crystal violet or methylene blue stained gel?

 

Thanks again.



#4 phage434

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Posted 08 February 2014 - 06:54 PM

You should (in my experience) have litle trouble amplifying 5.7 kb using a new polymerase such as Q5 or Phusion. Methylene blue will stain DNA effectively, but with much lower sensitivity than other dyes. You could use it, or use a blue light source instead of UV for a normal stain. Another option is to run a marker lane, which is exposed to UV, marked, and then used to cut a fragment from an adjacent, unexposed lane. But I stand by my previous post as the easiest way of making things work. You'll find that gel purification does not get rid of all of the background, especially if you are cutting only 45 bp from the parent plasmid. Qiagen PCR cleanup should be fine for any of the dyes.



#5 plasmamembrane

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Posted 09 February 2014 - 01:18 PM

I only have LongAmp ( NEB ) and Pfu enzyme. I have experience of amplifying 4.0Kb fragment with LongAmp, but I concern about the fidelity of polymerase. LongAmp Is only 2X more accurate than conventional polymerase. Should I sequence the vector after cloning?

By the way, could you please explain why 45bp short fragment cause a problem in gel elusion? Supercoiled and restricted plasmid run at difference pace and on gel , I think I can identify the

restricted band.

 

Thanks for your opinion.



#6 phage434

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Posted 09 February 2014 - 01:26 PM

You don't really care about possible mutations in your vector. If the mutation is serious, the vector won't work, and you will not have a colony. If the mutation is benign, then you get a colony, and you don't care about the vector sequence.

 

You don't get only supercoiled plasmid in a miniprep. You get a mixture of supercoiled, nicked, relaxed, linearized, and some tthings in-between. Some of those will run at nearly the same size as the fragment you are trying to isolate. You have a better chance of eliminating uncut or incompletely cut vector if the insert is large enough to make a singificant difference in the length of the cut fragment.






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