|
06/04/14
- cDNA synthesis: K562 sets ## - ##
cDNA synthesis: K562
- Use Carly's RNA from -80 °C
- Assume that K562 1.1 is +PcTF and K562 2.1 is control (will find out when QC plate for mCh RT-PCR is run)
- Will hold off on continuing U2OS for transiently transfected samples; will switch to KAH126 for further analyses
Measure RNA concentration
| Sample |
OD 260 |
260/280 |
ng/μL |
Vol. to use for cDNA synth.
|
| 1. K562 1.1 |
-0.006 |
1.968 |
-5.193 |
n/a
|
| 2. K562 2.1 |
-0.003 |
1.6 |
-2.724 |
n/a
|
- Conclusion: discard samples
- Do a new RNA extraction from TRIzol samples
RNA extraction
- Follow the TRIzol/ RNeasy protocol
- Use Carly's TRIzol samples K562 1, 2, 3, and 4
Measure RNA concentration
| Sample |
OD 260 |
260/280 |
ng/μL |
Vol. to use for cDNA synth.
|
| 1. K562 1 |
### |
### |
### |
### μL (### μg)
|
| 2. K562 2 |
### |
### |
### |
### μL (### μg)
|
| 2. K562 3 |
### |
### |
### |
### μL (### μg)
|
| 2. K562 4 |
### |
### |
### |
### μL (### μg)
|
oligo(dT) Primer annealing
| Reagent |
Vol
|
| total RNA (up to 2μg) |
up to 8 μL
|
| 50 μM oligo(dT) primer |
1.0
|
| 10 mM dNTP mix |
1.0
|
| DEPC-treated water |
= 8.0 - vol. total RNA
|
| |
10.0 μL
|
--> Incubate at 65°C/ 5 min. Immediately place on ice for 1 min.
cDNA synthesis mix
- K562 1.1 (E004)
- K562 1.1 (E005)
- K562 2.1 (C004)
- K562 2.1 (C005)
| Reagent |
Single rxn. |
Mix (x4)
|
| 10x RT buffer |
2.0 |
8.0
|
| 25 mM MgCl2 |
4.0 |
16.0
|
| 0.1 M DDT |
2.0 |
8.0
|
| RNaseOUT |
1.0 |
4.0
|
| SuperScript III RT |
1.0 |
4.0
|
| |
10.0 μL |
40.0 μL
|
--> Aliquot 10 μL of mix into 8-tube strip
--> Add annealing rxn. into each 10 μL aliquot
--> PCR machine: 50°C/ 50 min., 80°C/ 5 min., ice
--> Add 1.0 μL RNase H, incubate at 37°C/ 20 min.
--> Store at -20°C
| 
Pc-TF Genomics
