Making spike - in transcripts for mRNA normalization
Grow DH5alpha ecoli cells overnight with pSP64 poly ( A ) plasmid containing inserted spike - in sequence .
Miniprep plasmids , quantify .
You'll need a lot of plasmid .
Set up the digestion reaction in 50ul 1x EcoRI compatible buffer with ~ 3ug pSP64 - NSERT - poly ( A ) and 5ul EcoRI .
Incubate 2 hours each , 37C .
Save some plasmid for the gel below .
Pour a 1 % agarose gel and run ~ 100ng of digest and undigested plasmid , to check for cutting .
Assuming you observe complete digestion , clean up reactions using PCR clean up kit , pooling repeats of the same plasmid digest at this stage .
Quantify using nanodrop to have an idea for concentration .
Set up SP6 reaction master mix , from the kit reagents : 1x5xTxn optimized 5x buffer4ul 20ulDTT ( 100mM ) 2ul 10ulRecombinant RNasin0.75ul 3.75ul10mM rATP1ul 5ul10mM rUTP1ul 5ul10mM rCTP1ul 5ul10mM rGTP1ul 5ul4tUTP ( 10mM ) 2ul 10ulSP6 RNA Polymerase1ul 5ul .
Make new tubes of 6.25ul of linarized plasmid DNA .
For a kit positive control , dilute 1ul of the provided standard with 5.25ul of water .
Concentrations should be in the range of 100 - 200 ng / ul .
Add 13.75ul of SP6 reaction master mix to each tube of linearized DNA .
Incubate 1 - 2hr in 30 / 37C water bath .
Remove 2ul into PCR tubes for later gel .
To the rest , add 1ul of RQ1 RNase - Free DNase .
Incubate 15min at 37C .
To each reaction , add 40ul Ampure XP beads and mix well with pipette .
Let sit RT for 5min .
Collected beads to side in a magnetic rack .
Aspirated supernatant , all .
Add ~ 500ul 80 % etOH onto beads .
Let sit ~ 30 seconds , then aspirate off .
Add ~ 500ul 80 % etOH onto beads .
Let sit ~ 30 seconds , then aspirate off .
Make sure to get everything out of the bottom , small tip may help .
Let beads dry in the rack with a kimwipe over the open tubes , 10min RT .
Resuspend beads in 20ul hyclone H _ 2O .
Run ~ 3ul on 1 % agarose TAE gel for about 20min at 100V with the NEB 1kb ladder .
Visualize with your favorite dye ( we use sybrsafe now ) .
Quantify on qubit , dilute .
0.1ng / ul is a good working mix .
Benjy suggests using 4ng spikein ( each ) : 100ug total RNA .
Darach's had success with 1ng for ~ 5e7 cells ( for qPCR normalization ) .
