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Hello everyone this is my first post.
We are going to be running about 4 different EST libraries through the |Roche 454, and are trying to save some money and time by size selecting the fragments ourselves. Does anyone have some helpful tips / tricks?
Our current protocol involves:
1) Remove ribosomal RNA using invitrogen RiboMinus™ Eukaryote Kit
2) Selecting for poly adenylated RNA using Promega polyA+
3) Changing parameters for cDNA synthesis such as RNA concentration and extension time
4) Adding restriction enzymes after the above values have been optimized.
Does anyone have any other advice for this step.
If you do, your input would be greatly appreciated.
Thanks,
John W
We are going to be running about 4 different EST libraries through the |Roche 454, and are trying to save some money and time by size selecting the fragments ourselves. Does anyone have some helpful tips / tricks?
Our current protocol involves:
1) Remove ribosomal RNA using invitrogen RiboMinus™ Eukaryote Kit
2) Selecting for poly adenylated RNA using Promega polyA+
3) Changing parameters for cDNA synthesis such as RNA concentration and extension time
4) Adding restriction enzymes after the above values have been optimized.
Does anyone have any other advice for this step.
If you do, your input would be greatly appreciated.
Thanks,
John W
