User:Karmella Haynes/Notebook/Polycomb project/2010/04/29

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

04/29/10

  • ✓ RT-PCR: 4-day dox induction of Pc-ATF lines, mCh primer optimization
  • ✓ Senescence assay: expand KAH126-3, -4 cell cultures to 10 cm plates (maintenance med.)
  • ✓ Minipreps: FlpE (4)

RT-PCR

> Reaction 1: optimization of mCherry primers
--> cDNA templates

  1. KAH126-1 (1:1)
  2. KAH126-1 +dox (1:1)
  3. KAH126-1 (1:10,000)
  4. KAH126-1 +dox (1:10,000)
  5. FTRx (1:1)
  6. FTRx +dox (1:1)
  7. FTRx (1:10,000)
  8. FTRx +dox (1:10,000)

--> primers

  1. mCh f1/r1, 100 bp, #1-8
  2. mCh f2/r2, 100 bp, #9-16
Reagent Volume Master mix (x10) RT-PCR optimization 4/29/10
15 μL/lane; 1% agarose
cDNA 0.5 ---
10μM primers 1.0 10.0
2x GoTaq Green 10.0 100.0
dH2O 8.5 85.0
  20.0 μL

--> Aliquot 19.5 μL of each DNA mix to appro. wells
--> Add 0.5 cDNA to each well
--> PCR (96-well)

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
  • 72°C/ 3 min.
  • 4°C/ ∞

--> Result: primer pair one shows expected results at 1:10,000 dilution
--> Next PCR: full Pc-ATF sample set, plus gene target primers


Minipreps
> Check with E, P, and E/P digests

Reagent 1,4,7,10 2,5,8,11 3,6,9,12 Expected:
1. FlpE-1, E = 7160, 546
2. FlpE-1, P = 7706
3. FlpE-1, E/P = 5649, 1511, 546
4. FlpE-2, E = 7160, 546
5. FlpE-2, P = 7706
6. FlpE-2, E/P = 5649, 1511, 546
7. FlpE-3, E = 7160, 546
8. FlpE-3, P = 7706
9. FlpE-3, E/P = 5649, 1511, 546
10. FlpE-4, E = 7160, 546
11. FlpE-4, P = 7706
12. FlpE-4, E/P = 5649, 1511, 546
Hover name
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 2.0 2.0
10X buffer 1.5 1.5 1.5
EcoRI 1.0 --- 1.0
PstI --- 1.0 1.0
dH2O 10.5 10.5 9.5
 
 
 
 
 
 
 
 

15 μL --> 37°C/ ~15 min.


--> None look correct. Next time, do a "slow" transfection (heat shock, recovery).


Personal tools