8 replies to this topic

[wntiong]

i did 25ul pcr rxn against housekeeping gene, then loaded 10ul of pcr product into 3% gel. The pcr product is designed about 200bp. but, there is no pcr product at all after gel run. what could be the reason? thanks.

[Curtis]

have you checked your DNA/cDNA concentration? some times different PCR machines give different results, this happened to me

[nightingale]

the reason maybe in ur sample ( inhibitors ) or in ur PCR reaction..
have u run a positve control/previousely tested sample along with urs ?

Edited by nightingale, 15 January 2010 - 05:13 AM.

[wntiong]

nightingale, on Jan 15 2010, 09:12 PM, said:

the reason maybe in ur sample ( inhibitors ) or in ur PCR reaction..
have u run a positve control/previousely tested sample along with urs ?

nope i don't have the positive control. this was my first time run pcr after 2 years. but i did second run this morning, still no result at all, only some smears at the bottom which i think is not the pcr product. my second run i increased the template, primer concentration, no. of cycles and make sure everything added in sequence and correctly. still there is no result. i tried two different primers of two genes, still there is no result. need your advise how to troubleshoot.

[Vini]

wntiong, on Jan 18 2010, 03:04 PM, said:

nightingale, on Jan 15 2010, 09:12 PM, said:

the reason maybe in ur sample ( inhibitors ) or in ur PCR reaction..
have u run a positve control/previousely tested sample along with urs ?

nope i don't have the positive control. this was my first time run pcr after 2 years. but i did second run this morning, still no result at all, only some smears at the bottom which i think is not the pcr product. my second run i increased the template, primer concentration, no. of cycles and make sure everything added in sequence and correctly. still there is no result. i tried two different primers of two genes, still there is no result. need your advise how to troubleshoot.

why dont u try decreasing the annealing temp.???

[nightingale]

try to bring a previously tested positive sample and run it, let's see how it'll go..
this will tell u the source of your problem,
whether its in ur reaction or in ur sample..

if the previousely tested sample gave none, i would go and check my thermal cycler temperature,
if it REALLY measures 95C and 72C and the rest ... ( since u have tried the other options )
this is done with the help of the manufacturer, they will provide you with controls ( s.th like thermometers ) put in the wells of the PCR to check for the temperature...

in our lab we've encountered faint bands and after troubleshooting and excluding all the rest possible factors the problem was with the cycler itself, one raw in the PCR was malfunctional...

can u kindly tell, what is your sample ??


best of luck

[wntiong]

nightingale, on Jan 19 2010, 01:23 AM, said:

try to bring a previously tested positive sample and run it, let's see how it'll go..
this will tell u the source of your problem,
whether its in ur reaction or in ur sample..

if the previousely tested sample gave none, i would go and check my thermal cycler temperature,
if it REALLY measures 95C and 72C and the rest ... ( since u have tried the other options )
this is done with the help of the manufacturer, they will provide you with controls ( s.th like thermometers ) put in the wells of the PCR to check for the temperature...

in our lab we've encountered faint bands and after troubleshooting and excluding all the rest possible factors the problem was with the cycler itself, one raw in the PCR was malfunctional...

can u kindly tell, what is your sample ??


best of luck

thanks. my samples were total rna isolated from whole blood, been dnases treated.

[stylothecancer]

wntiong, on Jan 15 2010, 06:48 PM, said:

i did 25ul pcr rxn against housekeeping gene, then loaded 10ul of pcr product into 3% gel. The pcr product is designed about 200bp. but, there is no pcr product at all after gel run. what could be the reason? thanks.

Hi wntiong,

may i know how you kept your RNA sample? is it in TE buffer or just in the RNAse free water?

In my experience, I always had this experience when i used TE buffer to kept my RNA samples, which should be working fine, but not in my hand.  

So eventually, I always kept in RNA sample in RNAse free water.

[nightingale]

have u checked the integrity of ur RNA samples before running them on PCR ???
one simple method we used in our lab :-
is done by preparing a 0.7- 1.0 % agarose gel,
then load ur RNA samples along with EtBr
u should see 2 bands
18S and 28S

check this site  http://www.ambion.co...d=RNA integrity

good luck