help needed for labelling RNA at 3' ends

[tfitzwater at NEXSTAR.COM]

T4 RNA ligase labeling reaction 1:  adding [a(superscript: 32)P]-pCp to the 3'
end of RNA

Combine 30 pmoles of RNAwith 4 µL 10x T4 RNA ligase buffer, 4 µL DMSO, 9 µL of
3.33 pmol/µl [a(superscript: 32)P]-pCp (30 pmoles) and 80-100 Units T4 RNA
ligase in a 40 µL reaction.  Gently mix and incubate at 4-17°C at least 6 hours.
Incubation over the weekend is OK.  Do NOT incubate at 37°C unless a particular
RNA is difficult to label at 4°C.  Then try 37°C, or cycle in between 4°C and
37°C in a thermal cycler with slow ramping between temperatures, i.e. 4°C/30
min, 37°C ramp/30 min, 37°C/ 30 min, 4°C ramp/30 min for 3 to 30 cycles,
optional hold at 4°C.  Normally, 4°C incubations provide more efficient
labeling.  RNA sequences ending in a pyrimidines may be more difficult to label.
10x T4 RNA ligase buffer consists of 50 mM Tris-HCl, pH 7.8, 10 mM magnesium
chloride, 10 mM bME and 1 mM ATP.

Protocol modified from T. England and O. Uhlenbeck, 1978 Nature 275: 560.

Tim Fitzwater
NeXstar (a subsidiary of Gilead Sciences)

Jianxin Wang <jw24 at acsu.buffalo.edu> wrote:

>Hi, all, I need a protocol for labelling RNA at 3' ends. I will use T4
>RNA ligase and (5'-32p) pCp. Your input will be hightly appreciated!
>
>jw