shorter unspecific PCR product - (Sep/14/2005 )

Hi everybody,

I am trying to clone a 1650 bp fragment from chicken brain.When I use internal primer pairs that generates for example 900 or 700 bp fragments, cloning is succesful but when I try a primer pair that generates a longer fragment ( 1650 bp) it is not succesful anymore.Instead of 1650 bp, I see a band of 700 bp on agarose gel. I increased annealing temperature form 54 up to 57 C. but I still have the same problem !! Now I will try increasing Mg concentration.Is there anything else I can do? any comments will be appreciated:)

Thanks in advance...

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I think you can increase the temperature more 57 seems low... You shouldn't need a special taq to do 1650bp so is your extension time long enough?

Did you blast your 1650bp primers to the sequence, maybe there is a partial bindng site within your sequence that gives the smaller product... are the primer-primer interactions etc more significant with your 1650bp primers than your others?

Are there any special properties to the sequence ie: GC rich?

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Hi beccaf22,

My primer's Tms are 58.3 and 59.5.So I am not sure if I can increase the temperature more.May be I can try 58 or 59?

I increase extension time gradually from 3 min up to 6 min.Because my pcr works at an increasing extension time from 2 min to 4.5 min for my other smaller fragments( 700 and 900 bp) that I mentioned before. At the beginning I use this extension time( 2 to 4.5 ) for 1650 bp fragment too but I obtained 700 bp so I switched to the extenison time from 3 to 6 min. but I saw the same 700 bp band.

I align my primers to the sequence but they only align to the desired parts of the sequence. I don't know how to blast my primers to the sequence.When I write my sequence and one of my primer in FASTA format on NCBI's Blast program it only gives sequences similar to my sequence??It does not care about my primer I don't understand how to do it.

Thank you for the answer:)

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Hi, you have to use the blast 2 sequences command. At NCBI-BLAST this command is found in the section called "special" at the bottom. Put your primer in as one sequence and template as the other.

We also always blast primers to the genome we are working with and that can be done using the search for short nearly exact matches function (in the nucleotide section) and inputing the sequence of one primer at a time.

What you are looking for in the blast is at least 1-2 bp mismatch (more is better) at the 3' end of any similar sequence. Matches at the 3' end are the only ones important because of the 5'-3' directionality of polymerase. (ie: the polymerase uses the 3' end of the primer to initiate synthesis)

What about the G-C content of your template? BTW, I think you may be overdoing the extension time, typical rule is 1 minute extension per kb...

Also, you can go above the Tm for your primers. There may be a problem with decreased efficiency of the reaction, but usually primers can be used above their Tm if necessary (not too much of course!)

Good Luck! Let me know what happens...

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Hi:),

Thank you for the description:; I tried wrong one ( nucleotide-nucleotide blasting )

I did blasting finally but it says that there is only one sequence for my primer and it does not show any other similar sequence in my whole sequence.

For the extension time issue, when I tried from 2 to 4.5 only 700 bp band was bright and 900 was very weak but when I tried from 3 to 6 min 900 bp get brighter...So I thought my 1650 bp fragment would appear if I do from 3 to 6 but it didn't happen so. I could not see 1650 bp band but only 700 bp band:(

Additionally, my template is %52 GC rich...

Thank you so much

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What about the primer combinations, are you using the same 3' primer with different 5' primers or three competely different sets...

If same 3' maybe the 3' primer or your 1650bp 5' primer got contaminated by the 700bp 5' primer --- because it is a shorter product the amplification is more efficient for 700bp preventing the 1650bp from being amplified correctly???

You could test if the 3' primer is contaminated by a 5'primer by adding only the 3' primer to the reaction, if the 700bp primer is contaminating you will see bands... Not sure how you could test the 5' being contaminated with another 5'....

Did you blast to the genome (is it available?) maybe that 700bp fragment is another similar gene... Have you sequenced the 700 and 900bp products to make sure you got what you wanted?


HTH

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Hi:)

Sorry I could not answer you untill this time:(

hmm I use three primer combos: like this f2/r2 (900bp), f3/r3(700 bp)and f2/r3 (1650 bp).my whole seq. is 1970 bp long and so I didn't have a primer for only 300 bp fragment from beginning so I ordered a new primer for this region.(let's say f1:)) now I am trying these combos: f1/r2(1260 bp) and f1/r3(1970bp-whole seq.)but still I can not get what i want
.I see tha same patterns on agarose gel for both different combos???I attach the gel photo.first lane is f1/r2, second is f1/r3 .I use GC-MELT in these two pcr.the third lane is f1/r3 but w/o GC-MELT.

Yes I sequenced my two fragments 700 and 900 and I am pretty sure I got what I want

So what do you think about this issue:?

Thank you...

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Okey I finally managed to clone my entire sequence:)I did touchdown pcr and use higher Ta(from 62 to 60).So you are right that i can use higher Ta then my primers Tms.

Thank you...

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