3-25-2011 RT-PCR

Notebook: ZHEN TAO #10

Page: 002072

2ul sample

1ul WPRE-F primer

1ul WPRE-R primer

12.5 ul Mastermix (Roche)

8.5 ul H₂O

Total 25 ul.

1) 50C 2 min;

2) 95C 10 min;

3) 95C 15 sec;

4) 60C 1 min;

5) repeat step 3 and 4 for 40 times

tube number	1	2	3	4	5	6	7
position in plate	D3	D4	D5	D6	D7	D8	D9
sample	#13	#14	#13	#14	#13	#14	H2O
treatment	untreated		lysis and then DNAse		DNAse and then lysis
CT value	15.7	15.7	24.6	25.3	24.6	23.3	28.3

Treatment:

Untreated: 2ul virus mix with 18 ul TBS, and use 2 ul to do RT-PCR;

Lysis and then DNAse: 12 ul virus mix with 108 ul lysis buffer; RT, 10 min; add 2 ul 1M Tris pH8.0; add 1ul DNAse; RT, 30 min; 75C 10 min; use 2 ul to do RT-PCR;

DNAse then lysis: in 12 ul virus, add 1 ul DNAse, RT 30 min; 75C 10 min; add 108 ul lysis buffer; RT 10 min; use 2 ul to do RT-PCR.

--Ztao 14:48, 25 March 2011 (EST)