h*********g
发帖数: 18 | 1 I am using Ambion mMESSENGERmMACHINE Sp6 to make mRNA from plasmid. After
making mRNA, I run a simple agarose gel. There are two bands. One band is
not very close to the actual size (400bp smaller), there is also another
smaller and stronger band. Can I still use the mRNA? Why there are two bands
? Thanks. |
x**w
发帖数: 112 | 2 Did you purify mRNA before electrophoresis?
Un-incorporated NTP also could be detected by EB. |
h*********g
发帖数: 18 | 3 I did not purify mRNA. Could you tell me how to purify it? Using what kit?
Thanks!
【在 x**w 的大作中提到】
: Did you purify mRNA before electrophoresis?
: Un-incorporated NTP also could be detected by EB.
|
r********n
发帖数: 164 | 4 Did you use denature gel? RNA gel? Normal DNA gels may not give you good
estimation on the size. Purification I used RNAeasy kit from Qiagen, works
well. You could use classic pheno-chloroform extraction if you don't have
the kit. If your RNA coded product has fluorescence or very specific
phenotypes in your system maybe a direct functional verification is easier.
good luck |
c*********r
发帖数: 1312 | 5 mMESSENGER mMACHINE 说明书里提供了好像是三种纯化方法。试剂盒里也有一些纯化所
需的试剂。
【在 h*********g 的大作中提到】
: I did not purify mRNA. Could you tell me how to purify it? Using what kit?
: Thanks!
|
x**w
发帖数: 112 | 6 My experience is LiCl +isopropanol works perfect. |
h*********g
发帖数: 18 | 7 Thanks. I used normal DNA gel. I don't know why there two bands. Thanks.
【在 r********n 的大作中提到】
: Did you use denature gel? RNA gel? Normal DNA gels may not give you good
: estimation on the size. Purification I used RNAeasy kit from Qiagen, works
: well. You could use classic pheno-chloroform extraction if you don't have
: the kit. If your RNA coded product has fluorescence or very specific
: phenotypes in your system maybe a direct functional verification is easier.
: good luck
|
s********n
发帖数: 248 | 8 Did you digest the DNA template? Did you run a formaldehyde gel? Did you use
RNA ladder?
RNA runs at lower MW comparing to dsDNA; if you are not using RNA gel, your
RNA will be easily degraded. |
