Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

no insert found

cloning

Best Answer plasmamembrane, 02 March 2014 - 03:51 PM

After selecting colonies with colony PCR, 4 out of 36 colonies are positive for insert. I think the issue here is due to preferential annealing of primer-primer heterodimer into the vector thus generating more transformants in experimental plate than in control plate.

Thanks a lot for your opinions. I finally got my clone.

Go to the full post


  • Please log in to reply
14 replies to this topic

#1 plasmamembrane

plasmamembrane

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 21 February 2014 - 06:19 PM

I am trying to clone a 3.8Kb gene into 5.7Kb vector ( pFBDM )
Here is my protocol:

Vector preparation:
10ul of vector was diogested with two enzymes ( 1ul of XmaI and Kpn-I HF in CutSmart buffer , enzymes were from NEB as fresh stock ) at 37deg C for 3 hrs, then gel purify ( Qiagen gel elusion kit ).
Note: the DNA was not exposed to UV, I used another lane as standard and cut
at the corresponding site.

PCR:
cDNA was obtained by reverse transcription ( NEB RNase - reverse transcriptasem, primed by oligo-dT )
4 extra bases are added for efficient digestion. According to NEB manual, it will be sifficient for 100% activity.
amplification: LongAmp Taq ( NEB ). I saw a sharp band on agarose gel.
The PCR product was purified prior to restriction enzymes digestion ( Qiagen kit )
Enzymes digestion: same as vector, I purify the DNA again after digestion with Qiagen kit.

Ligation:
50ng of digested vector was used in ligation
vector:insert ratio: 1:2
ligation 16 degC O/N ( NEB ligase )
negative control: vector + ligase only, no insert

Plate:
negative control: 3 colonies
experimental plate: around 50 colonies

I picked 8 colonies and grew O/N at 37 deg C. Plasmid was purified using Qiagen kit. The plasmids preparations were digested with two enzymes, however, no insert found. If digested with single enzymes, the plasmid was of same size with original vector.

Thanks for your opinion.

#2 Ahrenhase

Ahrenhase

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 160 posts
10
Good

Posted 21 February 2014 - 06:56 PM

When you cut with your two enzymes, how big was the piece you cut out (i.e. base pairs between the 2 restriction sites).  If it wasn't much (<100), chances are that when you were cutting your cut band out of your gel, you got a little uncut plasmid in the mix and that's what you're seeing on the experimental plate.  



#3 plasmamembrane

plasmamembrane

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 21 February 2014 - 07:27 PM

I included a negative control ( cut vector only , no insert ), only 3 colonies yielded. The negative control essentially excluded the possibility.



#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,375 posts
227
Excellent

Posted 22 February 2014 - 09:52 AM

I'd like to know more about yoru reverse transcriptase reaction. You said you used an oligo dT primer -- did it have the XmaI or KpnI cut site? Or is there another primer used for the gene you are interested in. It sounds as if you have done many things correctly, but I'm uncertain about your final vector and insert cooncentration. I'd recommend next determining if your insert cut ends ligate efficiently. Do this by taking your PCR product, cutting with only one of the two enzymes, and ligating. You should get a double length fragment. Do this for both enzymes. You could also get rid of background by making your vector with PCR, which then allows you to digest the template with DpnI. I'm a little concerned about your transformation efficiency, because I would actually assume your background should be much higher after vector digestion and gel purification.



#5 plasmamembrane

plasmamembrane

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 22 February 2014 - 10:06 AM

I am using the oligo-dT primer not gene specific primer. I did not include RE in my RT primer.

 

I used 50ng of cut vector for ligation, insert/vector ratio was 2:1. The insert is 3.8Kb long; vector is 5.7Kb long.

 

The transformation efficiency of my competent cells is 10^8 CFU/ug plasmid. I tested it in positive control and worked fine.

Negative control yielded few colonies suggesting that self ligation is minimal; I am curious about why experimental plate contained so many background colonies?

Is it possible that bacteria delete the insert?

 

Thanks again.



#6 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,375 posts
227
Excellent

Posted 22 February 2014 - 10:15 AM

So, are you amplifying the insert with a different primer after the RT reaction? Where does the restriction site for your insert come from?

 

You quote a transformation efficiency number, but is that hoped-for, or measured? The ability to transform with prepared plasmid tells you essentially nothing about the required efficiency, unless it is done at the 10 pg of plasmid level.

 

For future reference, it is a bad idea to post to two fora. The conversation happening in the other forum is relevant and should be in one place. I disagree with pernesseblue about ligation concentration. Too high a vector and insert concentration leads to concatamers, which fail to transform. 20-50 ng of vector in a ligation is the sweet spot, along with equimolar insert.

Are you using quick ligation mix, or the normal ligation buffer?



#7 plasmamembrane

plasmamembrane

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 22 February 2014 - 10:23 AM

Oligo dT primer was used for reverse transcription. I did not use gene specific primer in RT.

I tested the transformation efficiency with 10pg of pUC19.

I used the regular T4 ligase and set up 16 deg C O/N ligation.

 

Thanks



#8 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,375 posts
227
Excellent

Posted 22 February 2014 - 10:58 AM

So, I don't understand how you expect to clone a fragment which does not have a restriction site at one end into a KpnI/XmaI digested vector. One end of  your insert wil have a poly T region, which won't clone.



#9 plasmamembrane

plasmamembrane

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 22 February 2014 - 08:17 PM

The RE sites are included in my PCR primer. So my PCR product contain the RE sites. The PCR product and vector were both digested with Kpn1 and Xma1.



#10 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,375 posts
227
Excellent

Posted 22 February 2014 - 08:23 PM

So, after RT reaction with an oligo dT primer, you do a PCR on the cDNA with a pair of primers, both of which have a restriction site. And this is what you see on a gel. Is that right?

If so, I repeat my suggestion about testing the ligation efficiency of single-enzyme cut versions of this PCR product.



#11 plasmamembrane

plasmamembrane

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 22 February 2014 - 08:30 PM

Yes, that's right.

Do you have any idea why a lot of background colonies on experimental plates while only a few colonies on negative control plate ( cut vector only,  no insert, with ligase )?

Thanks again.



#12 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,375 posts
227
Excellent

Posted 22 February 2014 - 08:54 PM

No, I have observed similar things before, and also did not understand the cause. When you get the DNA fragments going into the ligation correct, you will get a great many more correct clones. A possible issue not yet mentioned is that your insert is toxic in E. coli. You could try a low copy number plasmid. Outgrowth and incubation at a lower temperature might also help.



#13 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,375 posts
227
Excellent

Posted 23 February 2014 - 07:51 AM

Here's a possible answer to your question. Your PCR could be producing primer-dimers (hetero) which will have both cut sites. They will be cut and clone more efficiently than the longer correct fragment. They will be nearly invisible on a gel, since they are so short. Depending on how you are purifying your PCR reaction, some may be eliminated, but there will be lots more where they came from. Another possibility is that your insert has an unexpected restriction site close to one of the ends, which is leaving a short fragment that has correct ends, and a longer fragment that will be visible to a gel, but will not clone. Both of these could be checked by sequencing some of your 50 colonies "without" inserts to see if they really have short inserts.

For the primer-dimers, you could check your primers with the IDT tools. You might be able to fix this by adjusting PCR conditions (likely raising the anealing temperature). There isn't much to be done about an unexpected site, but it could be found by sequencing your PCR product.



#14 plasmamembrane

plasmamembrane

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 23 February 2014 - 08:37 AM

I analyzed the primers with IDT tools and found there're several possibilities of hetero primer-dimers. I think they might be the problem and can explain why low background on control plate whereas dozens of transformants on experimental plate. The short fragments generated by hetero primer dimers were clones into the vector and those inserts were not visible on gel analysis either single cut or double cut.

 

Since my PCR product was purified with Qiagen kit not with gel elusion, it is highly probable that the kit did not eliminate efficiently the primer dimers. One of my primer ( the one with XmaI RE site ) is over than 40 base pairs long. In my experience, Qiagen kit does not get rid of oligos or DNA fragments longer than 40mers efficiently. The reason I use such long primer is that

I added His tag as well as linker sequence that is crucial in my further purification procedure. I have no antibody to prepare the column.

 

 

I will run the PCR again and gel elute the product. I will let you know the result.

 

Thanks for your advice. 



#15 plasmamembrane

plasmamembrane

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 02 March 2014 - 03:51 PM   Best Answer

After selecting colonies with colony PCR, 4 out of 36 colonies are positive for insert. I think the issue here is due to preferential annealing of primer-primer heterodimer into the vector thus generating more transformants in experimental plate than in control plate.

Thanks a lot for your opinions. I finally got my clone.







Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.