CELL FREE SYSTEMS	> TRANSCRIPTION	> Set Up Reaction	> Order of Setup and Incubation
			> Reagents
			> Buffers
			> Sample Protocol
			> Transcription Reactions
			> Troubleshooting Transcription Kinetics

 

Sample Protocol

Get a printable version of this protocol (requires Adobe Acrobat) 

1. Experiment title and date.

    

2.  Specific purpose and questions addressed.

    

3.  Columns across the page: tube #; DNA to be transcribed; volume of DNA; volume of master mix; volume of H2O; total volume. Down the page, list numbered tubes and volumes for each component.

    

4.  

   Master T1 mix for 10 uL transcripts:

   WG		RRL
   CB5XSP6T1 (WG)	2.0 uL	CB5XSP6T1 (RRL)	2.0 uL
   4 NTP (5X) capped	2.0 uL	4 NTP (5X) uncapped	2.0 uL
   10X Gcap	1.0 uL
   0.1 M DTT	1.0 uL	0.1 M DTT	1.0 uL
   tRNA 10 mg/mL	0.2 uL	tRNA 10 mg/mL	0.2 uL
   SP6 pol 10 U/uL	0.4 uL	SP6 pol 10 U/uL	0.4 uL
   RNA guard 20 U/uL	0.4 uL	RNA guard 20 U/uL	0.4 uL
   add DNA and H2O --> 10 ul		add DNA and H2O --> 10ul

    

 

Sample Protocol - Transcription for RRL

10 uL reaction for 350uL reaction

MMT1

CB5X	2	70
4 NTP	2	70
0.1 M DTT	1	35
tRNA	0.2	7
SP6P	0.4	14
Rnasin	0.4	14
TOTAL= 6 uL

Tube	Plasmid	DNA	MMT'	H2O	Total Volume
1	A@0.5 mg/mL	2	6.0	2.0	10
2	B@ 1.0 mg/mL	1	6.0	3.0	10
3	C@ 1.0 mg/mL	2	12.0	6.0	20