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

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05/04/10

 * &#x2713; Minipreps: FlpE (4)
 * &#x2713; RT-PCR: Pc-ATF 4-day dox GAPDH controls
 * &#x2713; Hygro kill curve: check cells
 * &#x2713; Thaw cells: KAH126-1, 132-4, 132-6 (spin down in 5 mL FBS/ts; seed in 20% FBS/ts medium)
 * &#x2713; 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

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

--> 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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