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Degenerate primers


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#1 LTSAL

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Posted 27 January 2015 - 08:33 AM

Hi

 

I have to sets of primers,

 

the first set is suppose to amplify 242 bp product and the second set was suppose to amplify 1432bp.

 

My question is do I have to use the first set of the primers, amplify the 242bp , gel extract and reamplify the purified DNA to 1432bp?

 

The journal doesn't state it in detail however the final product is 1432bp



#2 CPRES

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Posted 27 January 2015 - 08:46 AM

If this is nested PCR, you will first amplify the bigger product (1432), and then the smaller one (242). You can't get bigger product out of smaller template!


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#3 LTSAL

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Posted 27 January 2015 - 09:22 AM

Hi

 

Cellcounter, that's what I thought too but some how the final product was 1432.

 

Attaching a part of the protocol

 

 

Attached Files


Edited by LTSAL, 27 January 2015 - 10:07 AM.


#4 Trof

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Posted 27 January 2015 - 12:54 PM

There is no way how you can reamplify 1432 bp from a 242 bp product.

 

You write the primers were degenerate.. so it means the sequence was not known... can you link the whole paper? It's impossible to guess if those two primer pair are actually related anyhow, or just a 1. and 2. try of something. And what was the purpose of it all, why 242 bp seems "bad" from the excerpt and 1432 bp is fine. It doesn't say you are suppose to use both, at least not this small part.


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#5 LTSAL

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Posted 28 January 2015 - 10:54 AM

Hi

 

Attached is the paper

Attached Files



#6 Trof

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Posted 28 January 2015 - 01:11 PM

So it's as I thought.

The paper describes a process of identification a gene with novel sequence, and they were tring to "fish it" with primers of similar organisms.You can use the BLAST Align to see yourself.

The Pseudomonas sp. AMS8 LipAMS8 gene GenBank accession is HQ162821.1 (mentioned in the paper).

Neither of the primer pair is completely specific.

 

The first pair has a single mismatch in reverse primer, the second pair has only match from 7-18th nucleotide of the primer (not so much specific, but the 3' end is matched so it may amplify). The first pair only amplified a small portion of the gene and they wanted it sequenced.

 

Now the question is what do you want. Amplify the complete gene?

 

I found there is a primer sequence, that amplifies the whole sequence and is correct, its' the one down in the table for the cloning, the pTrcHis TOPO TA (TA cloning doesn't need any overhangs, so its' just simply primers for whole gene). You may use the accession to check that it's really specific.

 

But also if you are amplifying not from a single colony, you may need to check for specifity of the primers in the genetic background you have.


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

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#7 LTSAL

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Posted 29 January 2015 - 11:33 AM

Hi there Trof

 

I want to amplify the lipase gene. Im just trying out this primer from the paper my supervisor wrote.  Im amplifying bacterial isolates which are proven to have lipase genes from protein assays and also qualitative analysis, however Im lost in this section.

 

Can you advise on how to create my own primers. I really don't know how to design primers.

 

I have the blast result for 16S rDNA sequencing though



#8 Trof

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Posted 29 January 2015 - 03:08 PM

Lipase gene form the same bacteria as mentioned in the paper or a different/unknown one?

 

If it's the same bacteria, then I already wrote that, use the pTrcHis TOPO TA primers from table in section 2.6. You've got conditions there as well. These should be specific to you sequence (check).


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 LTSAL

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Posted 31 January 2015 - 03:40 PM

Hi its from the same genus. Ok i will try to use the topo primers






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