I have problem with my PCR amplicon size. The desired size is 309 bp while I am getting band size of 550bp. I even change the primer My. Extract plasmid DNA for 3 times. Run PCR on both master mix and taq. Try out annealing from 48 to 64. Blast primers for so many times. But in each case I am getting band size of 550bp. What will be the logic behind this.
PCR reaction is 20ul.
M mix ..10ul
Primer ..0.5 each
Template..1ul
Water ..8 ul
94(3min)
94(30sec)
54(30 sec)
72( 1 min) 25x
72 (10min)
Gel pic is attached
Yes I isolated DNA for 3 different times.. all the time band size is 550 .. template used in PCR is 50ng/ul
Probably not what's going on, but just in case:
If you are performing the PCR on a plasmid with primers designed for plasmid sites flanking your insert (like m13), dont forget to add the length of plasmid DNA that is added to the original insert.
Well, it depends on what you want to do with the amplicon.
What is your final goal?
Again, assuming this is the case, the 550 bp band you have probably contains the product you want in it- only with additional plasmid DNA added on the beginning and the end .
if you have the plasmid map, check what is the distance between the two Primers you are using (on the empty plasmid/vector). Add that length of the insert- that should give you the length of the band you got in the PCR.
it you want no plasmid DNA, and to only amplify the original insert (309 bp), I would get new primers matching the exact sequence of the beginning and end of the insert.
Does this help?
One of my friend search my Gene by multiAlin software with selected primer.
He got different alignment site and product size.. in which one of them is 534bp size..
@Tzach Auman. Mulalin software is a bionformatics tool. Which show you the specificity of your primer.go through it
https://www.google.com.pk/url?sa=t&source=web&rct=j&url=http://www.sacs.ucsf.edu/cgi-bin/multalin.py&ved=0ahUKEwjv-uLbnKvVAhWDVxoKHYrvDVcQFggoMAE&usg=AFQjCNF8dAMW9d4Rc52fpxObDZcuMZsiQA
Though I am very late in reading this post but may be it comes handy for someone else. You can try one more thing look for the restriction enzyme sites at the end of your DNA Sequence in this case after amplification you can use Restriction enzymes and you will get two bands after running it on Gel. one band will be the DNA sequence that you were going to amplify and another band will be the plasmid in which you have your gene of interest.
hope it helps.
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