Preparation of template dna

Lithium chlorideprecipitation
Lithium Chloride (LiCl) precipitation is a convenient and effective way to remove unincorporated nucleotides and most proteins. Lithium chloride precipitation, however, does not precipitate transfer RNA and may not efficiently precipitate RNAs smaller than 300 nucleotides. Also, the concentration of RNA should be at least 0.1 μg/μL to assure efficient precipitation. To precipitate from reactions that are thought to have very low yields of RNA, do not dilute the transcription reaction with water prior to adding the LiCl Precipitation Solution in the step below.
1) Stop the reaction and precipitate the RNA by adding 30 μL Nuclease-free Water and 30 μL LiCl Precipitation Solution.
2) Mix thoroughly. Incubate for 30 minutes on ice.
3) Pellet the RNA by centrifugation at >10,000 x g for 15 minutes at 4°C.
4) Remove the supernatant with a pipette and gently rinse the pellet with 70% ethanol.
5) Remove the 70% ethanol with a pipette without disturbing the RNA pellet.
6) Allow pellet to dry, then resuspend in Nuclease-free Water, TE or other suitable buffer.
7) While usually unnecessary, steps 1-6 may be repeated a second time for even cleaner RNA.
8) Allow the pellet to dry, then resuspend in 25 μL of Nuclease-free Water or TE buffer.
9) Store frozen at -20°C or-70°C.
 
Phenol: chloroform extraction and isopropanol precipitation
It will remove all enzyme and most of the free nucleotides from High Yield T7 RNA Synthesis Kit reactions.
 1) Add 115 μL Nuclease-free Water and 15 μL 5 M ammonium acetate, and mix thoroughly.
2) Extract with an equal volume of phenol/chloroform (it can be water-saturated, buffer-saturated, or acidic), and then with an equal volume of chloroform. Recover aqueous phase and transfer to new tube.
3) Precipitate the RNA by adding 1 volume of isopropanol and mixing well.
4) Incubate for 15 minutes at -20°C.
5) Pellet the RNA by centrifugation at >10,000 x g for 15 minutes at 4°C.
6) Remove the supernatant with a pipette and gently rinse the pellet with 70% ethanol.
7) Remove the 70% ethanol with a pipette without disturbing the RNA pellet.
8) Allow pellet to dry, then resuspend in Nuclease-free Water, TE or other suitable buffer.
9) Allow the pellet to dry, then resuspend in 25 μL of Nuclease-free Water or TE buffer.
10) Store frozen at -20°C or -70°C.