User:Karmella Haynes/Notebook/Polycomb project/2010/05/04

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05/04/10

  • ✓ Minipreps: FlpE (4)
  • ✓ RT-PCR: Pc-ATF 4-day dox GAPDH controls
  • ✓ Hygro kill curve: check cells
  • ✓ Thaw cells: KAH126-1, 132-4, 132-6 (spin down in 5 mL FBS/ts; seed in 20% FBS/ts medium)
  • ✓ FlpE: streak plates using Bruno's glycerol stock (20 and 100 μg/mL Amp plates) Note: FlpE is maintained in Invitrogen TOP10 cells



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
E/P digests 05/04/10
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.


--> Conclusion: Failed again. Ask Bruno for glycerol stock. Perhaps FlpE is kept in non DH5α strain...?



RT-PCR

> Continued from 5/03/10: semi-qRT-PCR
--> Use 1:1000 dilution for GAPDH primers (10,000 might be too low)
--> cDNA templates

  1. KAH126-1
  2. KAH126-1(+dox)
  3. KAH127-4
  4. KAH127-4(+dox)
  5. KAH128-8
  6. KAH128-8(+dox)
  7. KAH129-4
  8. KAH129-4(+dox)
  9. KAH130-2
  10. KAH130-2(+dox)
  11. KAH131-9
  12. KAH131-9(+dox)
  13. KAH132-8
  14. KAH132-8(+dox)
  15. KAH133-1
  16. KAH133-1(+dox)
  17. FTRx
  18. FTRx(+dox)

--> Primers; cDNA dilution

  1. GAPDH(21A); 1:1,000
  2. GAPDH(21B); 1:1,000


Reagent Volume Primer mix (x20) cDNA mix (x3) RT-PCR 5/04/10
15 μL/lane; 2% agarose
cDNA 0.5 --- 1.5 (3x 0.5)
10μM primers 1.0 20.0 (20x 1.0) ---
2x GoTaq Green 10.0 200.0 (20x 10.0) ---
dH2O 8.5 80.0 (20x 4.0) 13.5 (3x 4.5)
  20.0 μL
 
 
 
 
 
 
 
 


--> GAPDH(21B) works well. Quantitate results using ImageJ.



Hygro kill curve
> Result: 200 μg/mL shows 100% cell death for Flp-in T-REx, KAH126-1, KAH130-2, and KAH132-8
> Take note for making hygro+ stables later



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