User:Karmella Haynes/Notebook/Polycomb project/2010/09/14

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09/14/10

  • ✓ Midiprep check: KpnI digests
  • ✓ ChIP/ Co-IP: wash & elute (see 9/13/10)
  • ✓ RT-PCR: p16 induction cDNA from 9/13/10



Midiprep check
> Check with KpnI digests

Reagent Volume Expected:
Backbone = 7489
Empty vector frag. = 913
1-3. KAH160/KAH015-1,2,3 = 2060
2. KAH161/KAH015 = 2060
4-6. KAH170/KAH015-1,2,3 = 1868
9/14/10 mp digest
15 μL/lane; 1% agarose
DNA(plasmid) 1.0 μL
10X buffer 1.5
KpnI 1.0
dH2O 11.5
  15 μL --> 37°C/ ~15 min.
 
 
 
 

RT-PCR
> Use cDNA from "p16 time course" samples
> Same PCR conditions as p16 (see 8/26/10)
> Templates + Primers:

  1. p16 time course (1-16) + p16 7C (0.5 uL cDNA)
  2. p16 time course (1-16) + mCherry f1/r1 (1.0 uL 1:1000 cDNA dln.)
  3. p16 time course (1-16) + GAPDH 21A (0.5 uL 1:1000 cDNA dln.)


Reagent 1-16, MM (x17)</u> 17-32, MM (x17)</u> 33-48, MM (x17)</u>
cDNA --- (0.5/ rxn) --- (1.0/ rxn) --- (0.5/rxn)
10 μM primers 17.0 (p16 7C) 17.0 (mCh f1/r1) 17.0 (GAPDH 21A)
2x GoTaq Green 170.0 170.0 170.0
dH2O 144.5 136.0 144.5
  20.0 μL/rxn


--> Aliquot MM's: 19.5 for 1-16, 19.0 for 17-32, 19.5 for 33-48
--> Add cDNA
--> PCR (96-well)*

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
  • 72°C/ 3 min.
  • 4°C/ ∞

(*Same as p16INK PCR, 8/26/10)

> Conclusions: No signal for p16. Re-make cDNA with 5 ug RNA. Non-specific mCherry, use less template next time.



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