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No PCR product detected in agraose gel - (Jul/17/2006 )

I am currently doing RT-PCR and PCR for Rho A gene (size of ~570bp) from mouse embryo..

My protocol for RT-PCR is

1)Add 1ul of RNA + 3.5ul of DEPC treated water
2)Incubate at 75C for 5 mins and cooled to 42C for 5 mins
3) Add enzyme cocktail of

- 1ul 0.1M DTT
- 2ul 5X RT buffer
- 0.5ul 10mM dNTP mix
- 0.5ul RNAse inhibitor
-0.5ul oligo(dT)15 500ug/ul
- 1ul superscript 3
==> Total: 10ul

4) Incubate 42C for 1.5grs
5) Place on ice to terminate first strand synthesis

I then proceeded with PCR with the added reagents and protocol of

- 3ul 10X PCR buffer
- 0.6ul dNTP mix
- 0.6ul primer mix
- 0.24ul Taq Polyermase
- 1ul RT-reaction product
- 24.56ul millipore water
==> Total 30ul

94C - 6mins

94C - 30s
52C - 30s
72C - 30s
(above 3 steps for 35 cycles)

72C -10mins

Lastly extraction of the product by agarose gel electrophoresis using 1% gel at 140V for 50 mins using Promega 100bp ladder..

However I didn't get any bands at all after the gel under UV light..the protocols were given to me by my supervisor but she wasn't able to help me troubleshoot the problems..can someone help me? My fren suggested that the pore sizes of the gel were too big for my gene..possible? Thanks!!!!


Are you sure, that you have any RNA at ther very beginning of the procedure? If not, check this on a gel.... huh.gif


Did you see the ladder on the gel?


Ya the RNA is newly ordered so I assume it's in good condition..yes I can see the ladder but not very clear..


QUOTE (xiaoyuer @ Jul 17 2006, 04:47 PM)
Ya the RNA is newly ordered so I assume it's in good condition..yes I can see the ladder but not very clear..

newly ordered is fine, but still...measure some in the photometer if you don't have enough RNA do look at on a gel, or if the RNA is small-sized.
What kind of gel do you run?


Agarose...going to try using a higher percentage of gel with lower volts tmr..if that doesn't work I will ask for help again T_T


What concentration of ethidium bromide are you using?


I am using 10mg/ml ethidium bromide...have already tried using 1.6% gel at 100V for 1 partner who is using the same RT product but expressing a different gene (hence different PCR product of 990bp) has bands in the same gel that I am using...I do not have a single band at all...

Kinda loss at the moment..I'm not well verse in molecular biology..planning to order new set of primers or meddle with the temp..don't think it's the gel problem..?...hopefully can have some suggestions!!