Arichard:Reagents/celseq primers

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Revision as of 22:11, 14 February 2013 by >Andrew
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CEL-Seq RT primer layout[edit]

Stock layout[edit]

  • Barcoded RT primers are in 48 wells of a 92 well plate at 200 uM each, in the following layout:
Barcode number 1 2 3 4 5 6 7 8 9 10 11 12
A 1 2 3 4 5 6 7 8 9 10 11 12
B 13 14 15 16 17 18 19 20 21 22 23 24
C 25 26 27 28 29 30 31 32 33 34 35 36
D 37 38 39 40 41 42 43 44 45 46 47 48
E Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty
F Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty
G Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty
H Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty
  • Serial dilutions are necessary to dilute the RT primers to the correct concentration.

Diluted primer layout[edit]

  1. Dilute primers to 32 pmol/ul, 3.2 pmol/ul, and 0.32 pmol/ul, depending on input (Note that each primer must be diluted separately for each sample):
    1. Add 16 ul of 100 pmol/ul primer to 84 ul H2O --> 100 ul 32 pmol/ul primer. Use 0.5 ul of this per 1 ng total RNA.
    2. Add 10 ul of 32 pmol/ul primer to 90 ul H2O --> 100 ul 3.2 pmol/ul primer. Use 0.5 ul of this per 100 pg total RNA.
    3. Add 10 ul of 3.2 pmol/ul primer to 90 ul H2O --> 100 ul 0.32 pmol/ul primer. Use 0.5 ul of this per 10 pg total RNA.
100 ul/well 1 2 3 4 5 6 7 8 9 10 11 12
Conc 32 pmol/ul 3.2 pmol/ul 0.32 pmol/ul 32 pmol/ul 3.2 pmol/ul 0.32 pmol/ul 32 pmol/ul 3.2 pmol/ul 0.32 pmol/ul 32 pmol/ul 3.2 pmol/ul 0.32 pmol/ul
A #1 #1 #1 #2 #2 #2 #3 #3 #3 #4 #4 #4
B #5 #5 #5 #6 #6 #6 #7 #7 #7 #8 #8 #8
C #9 #9 #9 #10 #10 #10 #11 #11 #11 #12 #12 #12
D #13 #13 #13 #14 #14 #14 #15 #15 #15 #16 #16 #16
E #17 #17 #17 #18 #18 #18 #19 #19 #19 #20 #20 #20
F #21 #21 #21 #22 #22 #22 #23 #23 #23 #24 #24 #24
G #25 #25 #25 #26 #26 #26 #27 #27 #27 #28 #28 #28
H #29 #29 #29 #30 #30 #30 #31 #31 #31 #32 #32 #32