do I get right PCR product - (Dec/29/2007 )
Dear Forumers,
I did a RT-PCR from mice tissue, ORF of my cDNA is 378bp, after I did a electrophoresis, I got a very bright band near 400bp band which maybe the right product. the problem is this bright band is too wide from near 400 to near 500, approximate 2 times as wide as marker.
I cannot figure out what's wrong with my PCR? I appreciate your help
How much PCR product do you load on the gel. You can reduce that run slightly longer so that 400bp and 500bp bands of ladder clearly separate. If you are still getting thick(saturated?) band then may be you can reduce the number of cycles in your PCR or reduce the template.
Overloading a gel will result in very broad bands. You probably have the correct result. Check by diluting your PCR product 10x and 100x and loading the gel with the diluted DNA and your marker lane.
I load 5μl PCR product and 1μl 5X loading buffer on the gel. Marker I is 6μl which is 100bp 200bp 300bp 400bp 500bp
and the number of cycles in my PCR is 50
Calvin, do you think I got a nonspecific amplification band near my gene of interest which is 378bp?
dear phage434 I diluted my PCR product to 1/10 1/50 1/100 1/500 conc. but I get the same result, I can see band on 1/10 and 1/50 conc in reduced brightness. 1/100 and 1/500 generate nothing
so overloading may not be the reason
There is never a reason to do 50 cycles. 35 is more than enough even for very dilute samples, and 40 is the absolute maximum ever. You are simply asking to produce garbage by cycling longer.
Thank You very much indeed:)
I will run PCR again
I load 5μl PCR product and 1μl 5X loading buffer on the gel. Marker I is 6μl which is 100bp 200bp 300bp 400bp 500bp
and the number of cycles in my PCR is 50
Calvin, do you think I got a nonspecific amplification band near my gene of interest which is 378bp?
I dont think the problem is with the non-specificity. As 378bp is very near to 400 it could be difficult to make out due to huge amount of DNA. You can try as suggested by phage434 to reduce PCR cycles. In-fact 30 cycles are sufficient and maximum of 35 if needed. Try increasing the gel conc (2%) for better resolution. May be then you can also upload both the pictures here so that it will help to see if it is due to over loading or something else. Good luck.
I load 5μl PCR product and 1μl 5X loading buffer on the gel. Marker I is 6μl which is 100bp 200bp 300bp 400bp 500bp
and the number of cycles in my PCR is 50
Calvin, do you think I got a nonspecific amplification band near my gene of interest which is 378bp?
I dont think the problem is with the non-specificity. As 378bp is very near to 400 it could be difficult to make out due to huge amount of DNA. You can try as suggested by phage434 to reduce PCR cycles. In-fact 30 cycles are sufficient and maximum of 35 if needed. Try increasing the gel conc (2%) for better resolution. May be then you can also upload both the pictures here so that it will help to see if it is due to over loading or something else. Good luck.
Calvin, here is my picture. Lane 1 is marker Lane 2 is DNA of original conc and Lane 3-6 are DNA diluted at various conc(shown in picture)
the gel cons is 2% agarose, Goldview (an alternative of EB)
the width of PCR product are the same at different conc
This is my first time do PCR
I appreciate your help:)
Wish U a fruitful 2008
well i would try the following :
run less product (1/10 and 1/540 dilutions are good)
run on a more concentrated agarose gel (enhance resolution at this size)
run at lower voltage (enhance thickness of the band
but it seems to me the product you're anlyzing may be the correct one plus an other non specific one
for next experiment, run less numbner of cyles (enhance non specificity)