a problem in RACE

[mia]

HELLO,

i hope somebody could help me. i am doing race pcr now and i have smear. i tried the troubleshooting guide of the kit but no way.i use clonthech bd kit. i do the same protocol of the kit. should i make dnase digestion for my rna. i used the ribopure ambion kit for rna extraction.
thank you

[moleculardude]

Hello Mia,
welcome to the forums!

have you checked your RNA on an agarose gel?

Moleculardude

[mia]

hi

thank you for your reply.
no, i didn't check my RNA on agarose gel because i usually had a low concentration (2-5 ug). do u think i have to check it?
thank you

[admin]

Hello Mia,

is that total RNA of 2ug - 5ug?

What is your concentration per microliter?

welcome to the forum!

[mia]

hi

my RNA is only 200ng/ul. so i think it is very poor, thus the whole concentration is 2-5ug.

thank you

[admin]

Hello Mia,
I think that sounds right. If you dissolved your RNA in a large volume (or eluted in 40-50ul using the Qiagen columns) then the concentration gets low.

How are you obtaining the RNA? Make sure your RNA is not degraded first as your tissue/cells RNA may be getting degraded. Try RNAse inhibitors as well in your samples to prevent smearing.

Also how much RNA are you adding to your RACE?

[mia]

Hi Admin,

thank you for your reply. i used 4 ul for the RACE (800ng). i didn't check my RNA by agarose.
but i checked the cDNA by PCR only using my GSPs and they give one clear band.
i used RNAse inhibitor to avoid degradation of the RNA in the RACE reaction.