I extracted DNA from bacteria by using extraction Kit, by when I run the DNA on agarose gel no DNA band detected, but when I did the PCR reaction I got good result. And when I run it on agarose gel I got good bands?
Yes, artur sir is right. PCR gives u amplifications so eventually u get ab increased concentration of the template DNA. May be there is a band and the visibility is poor, sir.
I run it twice and stained it by using both Gelred and t EtBr. the PCR was for 16s r RNA gene and it gave me a sharp band . is my PCR ok? and if is good for sequencing or not?
Mushtaq sir, I believe that your PCR is good if u have a single sharp band at the required bp. And if the marker band and ur 16s rRNa band coincides at the appropriate molecular weight, it is a go for sequencing. Hope I am right.
thank you santanu and arthur. i will sent it for sequencing and i hop ....
Hi,
While feeling lucky getting positive bands after PCR, check your that particular PCR with no template control. If you see a band in that reaction it is invalid. This helps you a lot conforming your work.
for 16SrRNA gene amplification you do not need high amount of template of genomic DNA. because that gene is a multi copy gene and u can amplify the gene in pcr with lot f amplicons. by doing pcr you increase the copy number of the gene, which is easily visualize in the gel electrophoresis. but be careful not to contaminate yourself and the lab environmnt from your amplicone which result contamination in your next pcr. therefore do a negative or control pcr along with your pcr.if control sample contain same size band u cannot conclude that u have amplify the exact gene u want.good luck.
thank you nilmini
but i do not understand "but be careful not to contaminate yourself and the lab environmnt from your amplicone which result contamination in your next pcr. therefore do a negative or control pcr along with your pcr.if control sample contain same size band u cannot conclude that u have amplify the exact gene u want."
when I do PCR with 2 primers for 1 gene Even in more than 1 bacteria all products wil be same size.
Hi Mushtaq,
Yes, you can get same-sized PCR products from PCR tubes you add their genomic DNA in.
The 'negative control' tube is set up without any DNA template. If negative control is amplified, what do you think what has happened? An 'un-intended' DNA template accidentally 'falls' into the tube. We call this 'contamination'. Since even the negative control can be amplified with an expected PCR product, can you trust the PCR bands from the other 'sample' tubes? You don't. Because they may also result from the same 'contaminant DNA template', not from your intended gDNA samples. So, set up a negative control in PCR experiment is very important.
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