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
| 
Project name
Main project page
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