q**********0
发帖数: 335 | 1 Recenly I use T3 megascript do in vitro transcription. The results is always
bad, the OD260/280 is about 1.53. A big white pellet can be seen after
isopropanol precipitation and RNA concentratin measure is about 1.0 ug/ul.
However, when run a RNA gel, the band is very faint even using 5 uL. I don't
know why? Maybe there is protein contamination? or RNAse digestion? Please
help. Thanks a lot! |
a****o
发帖数: 1786 | 2 the pellet may be unincorporated NTPs. try to use more template DNA. |
a****k
发帖数: 1130 | 3 We normally do overnight reaction for in vitro transcription. Make sure your
DNA template is good.
always
't
Please
【在 q**********0 的大作中提到】
: Recenly I use T3 megascript do in vitro transcription. The results is always
: bad, the OD260/280 is about 1.53. A big white pellet can be seen after
: isopropanol precipitation and RNA concentratin measure is about 1.0 ug/ul.
: However, when run a RNA gel, the band is very faint even using 5 uL. I don't
: know why? Maybe there is protein contamination? or RNAse digestion? Please
: help. Thanks a lot!
|
q**********0
发帖数: 335 | 4 Thanks. Sounds reasonable.
【在 a****o 的大作中提到】
: the pellet may be unincorporated NTPs. try to use more template DNA.
|
q**********0
发帖数: 335 | 5 Thanks. I used 5 hours, it may not long enough.
your
【在 a****k 的大作中提到】
: We normally do overnight reaction for in vitro transcription. Make sure your
: DNA template is good.
:
: always
: 't
: Please
|
a****o
发帖数: 1786 | 6 5hr should be long enough.
I think you need to make sure the plasmid is correct first.
【在 q**********0 的大作中提到】
: Thanks. I used 5 hours, it may not long enough.
:
: your
|
