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problem with poly A adding

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#1 tantao



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Posted 22 June 2011 - 02:09 AM

Dear everyone,
I use mMESSAGE mMACHINE (AM1344) to in vitro transcription two plasmids ( borh genes are cloned into pcDNA3.1(+) vector). After in vitro transcription (20ul reaction and the plasmid templates are both 1ug) , I use Poly(A) tailing kit to add poly A to the IVT products. Before the adding Poly(A), I take 0.5ul of IVT products(sample1 and sample2) as enzyme minus control, and after adding Poly(A) I take 1ul reaction products form 100ul products, then run non-denaturing-gel. I also quatiation the last products after recovery of RNA by phenol: chloroform ectraction. The results are as follow:
1. before adding the E-PAP Poly A enzyme, I use 0.5ul products from 96ul reaction buffer to run non-denaturing-gel. After adding he adding Poly(A), I use 1ul products to run non-denaturing-gel. However I can not see band in sample 1 before or after adding Poly(A). I can see band before or after
adding Poly(A). The marker is 100bp and the 500bp is 150ng.(figure1)

2. After recovery of RNA by phenol: chloroform ectraction, the RNA is ressolved in 20ul DEPC water I quatiation the last products by UV light absorbance. The concentartion of sample 1 is 5200ug/ml and sample 2 is 5300ug/ml. OD260/280=1.9. However I can see weak band in sample 1, and sample 2 is not like 5300ug/ml, after I take 1 ul to run non-denaturing-gel(figure2).
I do not know what is wrong?

Attached Thumbnails

  • figure 1.JPG
  • figure2.JPG

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