PCR problem

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debanjan.pubiot
debanjan.pubiot's picture
PCR problem

hii
this is debanjan.....i m trying to clone amylase gene from unknown mareine bacteria by pcr amplifiaction method...the primers what ever i have designed it  was reported earlier .....during pcr amplifiaction i am not getting the gene size of my interest what may be the cause....i will be very greatful if some one can help me solving the problem?
 

Shubhangi
Shubhangi's picture
Since your bacterial is

Since your bacterial is unkown its full genome is also not known. There might me a chance that primers are targeting some other strech of DNA for amplification. It might be possible that primers are joing somewhere in the midway of gene.
Sequence your amplified portion. See if it is part of amylase gene only.  If so you may try to using a different set of primers.

R Bishop
R Bishop's picture
 How much different is your

 How much different is your  PCR product? Is it a few base pairs of off by 100s?
There are a couple of ways to go from here depending on the difference in size.
To start, I agree with Shubhangi, design a new primer set for the gene.
Alternatively, you can design a set of nested primers that might amplify a shorter part of the internal sequence. You will need to gel purify the band, then perform PCR with the nested primers on the purified DNA piece.  This will get rid of genomic contamination etc.
Finally, you could just sub-clone the PCR product into a PCR cloning vector and sequence in one direction to see if its the correct gene. 
 
Good luck
 
Rus

Ivan Delgado
Ivan Delgado's picture
Hi Debanjan,

Hi Debanjan,
This is one of my favorite kinds of experiments: discovering a new gene. The amylase gene is a conserved gene so you should be able to identify regions that are identical across a large component of microbes. To identify a region that is highly conserved, a good rule of thumb is to align a bunch of DNA sequences from various microbes (related ones as well as others that are not as related). Hopefully the primers you are using where designed this way. Even if these primers were designed this way, they are nothing more than a best guess since you do not know the sequence of your new gene, so it is a good idea to try more primers sets. In addition to multiple primers sets, you also want to run your PCR under different conditions (if you have a gradient PCR machine that would be ideal). In short: get to know well the DNA region you are designing primers for. If you have a good feel for how much this region varies across many bacteria you will increase your chances of fishing out this gene from this unknown bacteria.
I am not sure if the structure of the amylase gene has changed much during bacterial evolution but my guess is that it has not. So your concern about the size of your PCR product is likely well founded (assuming that the difference is in the order of >100s of bp and not <100 bp). You may be amplifying a non-specific region, as suggested by Shubhangi and re-stated by Rusty. Ways of getting around this (figuring out what is going on) are: 1. test more primer sets, 2. different PCR conditions, and 3. sequencing the PCR product you are getting. 
Good luck

Jason King
Jason King's picture
How large are you expecting

How large are you expecting the PCR product to be and how large is the product you have. If what you have is shorter but appears to be a single band then maybe you need to extend your polymerization times. 1 minute per 1Kb is, I think, the rule of thumb. Also extend the melting and annealing times if they are very short (ie. less than 30 seconds).

smallcat227
smallcat227's picture
Sequence your product and see

Sequence your product and see whether it is part of your target gene or other unrelated gene, if the former, prolong the extending time in the PCR reaction; or you should redesign your primers.