User:Karmella Haynes/Notebook/Polycomb project/2010/07/23

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07/23/10

  • ✓ RNA prep: for Nanostring assay (cDNA synth. on Monday)



RNA prep
> Use Qiagen QIAshredder and RNeasy kit (no BME added to lysis buffer)
> Elute & re-elute with 30 μL RNase-free H2O

Sample OD260 260/280 ng/μL μL = 2 ug for cDNA*
KAH126-1 17.960 2.09 718.41 2.8
KAH126-1 dox 13.725 2.09 548.99 3.6
KAH128-8.3 15.208 2.10 608.31 3.3
KAH128-8.3 dox 7.159 2.03 286.35 7.0
KAH129-4 16.627 2.12 665.06 3.0
KAH129-4 dox 8.195 2.05 327.81 6.1
KAH132-8 14.053 2.10 562.13 3.6
KAH132-8 dox 9.859 2.02 394.37 5.1
KAH154-2 17.894 2.11 715.76 2.8
KAH154-2 dox 15.636 2.13 625.45 3.2
KAH156-5 22.152 2.10 886.09 2.3
KAH156-5 dox 16.320 2.10 652.80 3.1
KAH157-1 17.876 2.09 715.04 2.8
KAH157-1 dox 13.477 2.13 539.08 3.7
KAH158-4 17.331 2.10 693.22 2.9
KAH158-4 dox 12.785 2.14 511.39 3.9
KAH159-2 17.673 2.13 706.90 2.8
KAH159-2 dox 9.722 2.02 388.90 5.1
FTRx 18.622 2.10 744.88 2.7
FTRx dox 18.194 2.12 727.78 2.7

--> Store at -80°C *Note: use some for cDNA for RT-PCR confirmation of Nanostring results


7/26/10
> oligo(dT) Primer annealing

Reagent Vol
total RNA (up to 2μg) up to 8 μL
50μM oligo(dT) primer 1.0
10 mM dNTP mix 1.0
DEPC-treated water ---
  10.0 μL --> 65°C/ 5 min.; ice/ 1 min.


> cDNA synthesis mix
--> 10 reactions total

Reagent Vol Mix (x20)
10x RT buffer 2.0 40.0
25 mM MgCl2 4.0 80.0
0.1 M DDT 2.0 40.0
RNaseOUT 1.0 20.0
SuperScript III RT 1.0 20.0
  10.0 μL 200.0 μL

--> Add 10 μL mix to each annealing rxn.
--> 50°C/ 50 min., 80°C/ 5 min., ice
--> Add 0.8 μL RNase H, 37°C/ 20 min.
--> Store at -20°C