In Vitro Transcription/Translation
A.) Digest plasmid:
1.) Linearize plasmid with appropriate enzyme, example:
Eco RI Myo D Not I
--------------/-----------------------/--------------
T3-Pol> < T7-Pol
vector 6 ug ul
enzyme 2 ul
buffer 4.4 ul
H2O 35.2 ul
----------------------
50 uluse appropriate RNA-Polymerase (T3 or T7). Cut with Not I and use T3-Polymerase.
2.) Digest for 2 hours, then fill up to 100 ul TE.
3.) PH/Chloro 1x
4.) Chloro/IAA 1x
5.) NaAc ppt.
6.) Resuspend pellet in 21 ul RNAse free TE. Quantitate 1 ul on agarose gel.
B.) In vitro transcription: Use stratagene kit.
1.) 20 ul 5x transcription buffer
1.33 ul 0.75 M DTT (Promega)
2.5 ul RNAsin (Promega) 1 U/ul
20 ul XTP 2.5 mM resuspend in DEPC
20 ul linearized plasmid 2 ug
1 ul T7 RNA Polymerase (10 - 20 )
(2.5 ul T3 RNA Polymerase 40 U, Promega)
35.17 ul H2O DEPC
-------------------------------------------------------------------
100 ulcan use 1/2 of transcription mix, but still use 2 ug of plasmid ans same amount of polymerase.
2.) For T7 Pol. incubate for 1 hour at 37 C.
For T3 Pol. incubate for 2 hours at 41 C. Boost after one 1 hour with 2.5 ul T3 Pol.3.) PH/Chloro 1x
4.) Chloro/IAA 1x
5.) NaAc ppt. (all has to be RNAse free)
6.) Resuspend pellet in 20 - 24 ul H2O DEPC
7.) Aliquot RNA as needed and store at -70 C.
C.) In vitro translation:
1.) 35 ul Reticulocyte lysate
1 ul RNAsin (Promega)
1 ul 1 mM Amino Acid Mix (minus Methionine)
1-8 ul RNA
5 ul S35 Methionine
7 ul H2O to 50 ul total2.) Incubate in 30 C for 1 hour. (Can vary amount of RNA, according to best efficency of translation). It is possible to use 1/2 of the vol. too.
» Top