Contamination in sequencing sample

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Maddy
Maddy's picture
Contamination in sequencing sample

Dear All

I am Grad student, never done cloning before. Please help me with My prob.

I always get  a comment that my sequencing samples (T vector clone) are contaminated.

 Can anyone tell me what does that mean?

Ivan Delgado
Ivan Delgado's picture
 

 
It depends on what "contaminated" means. If you are sequencing your clones and you are getting multiple peaks per nucleotide (i.e. instead of getting a clear A peak, you are getting both an A and a T peak at the same location), then it is possible that you purified more than one sequence with the clone you isolated.

The best way to get around this is to always isolate multiple clones and sequence them. Getting "contaminated" clones is rare, so if you sequence a few at the same time you should be able to get at least one clone that is not contaminated.

Maddy
Maddy's picture
Thank you Ivan

Thank you Ivan

As U said, I have always been isolating multiple clones ad sequencing them.

But most of the clones are contaminated and only few are sequenced, which do not match the desired

sequence.

I understand that it is because of non specific amplifcation, of the desired band size

i have redesigned the primers twice already.

How else could I get around with this?

Ivan Delgado
Ivan Delgado's picture
 

 
One alternative, which requires additional work, is to design your primers with restriction enzyme sites in them. Once you run the PCR and purify the fragments, you then restriction digest the fragments before cloning them into a vector. This way you are introducing an additional selection step (restriction digest), so you should not have no contaminants among your clones. 

It is definitely more work, and you need to generate enough PCR fragments, but in a situation such as yours you need to do something extra to get rid of the contaminants. This is one way to do it.

Good luck

Maddy
Maddy's picture
Ivan, Thank you for your time

Ivan, Thank you for your time.

But  I have always designed primers with restriction sites.

And confirm the clones by restriction digestion.

Inspite of  this, I have been recieving the contamination comment. That is why I dont understand.

I spoke to the genotech company ( where sequencing was done), regarding this.

They said, contamination can be because of two reasons: one, different fragments in the vector.

two, Some looping structure being formed after their PCR.

They conducted the sequencing following denaturing to confirm for the looping ( yesterday).

This time they were able to sequence, they said. I am yet to recieve the sequence.  I am just waiting for

it!

Ivan Delgado
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I hope your sequence works.

I hope your sequence works. If it does not, another alternative is to simply synthesize the whole fragment. If your fragment is not too large (less than 500 bp), you can get it full synthesized, and already in a vector, at a number of companies. 

Cheers

Maddy
Maddy's picture
I hope it works.

I hope it works.

The fragment is about 800bp,

I am not sure if it works economical for the lab to synthesize.

Anyways, I wil keep that option open.

Thank you Ivan

Maddy
Maddy's picture
Hi Ivan

Hi Ivan

I Got the gene clone, that I had been struggling from past 3 months ( from My previous posts).

I have redesigned the primers atleast four times, have tried routine PCR, touch down PCR with & without hot start. I even tried Qiagen's one step RTPCR kit but they never worked.

This time i just changed the cDNA source. I was using CHO DG44 earlier, this time I used CHO k1. For the very first time it worked.

I hope this piece of information may be useful for others who follow the posts