Experiments to establish the protocol for hybridization to Research Genetics filters. We used Stratagene Universal Target total RNA to make P33 labeled probe according to the manufacturer. Experiments 1 and 2 explored the amount of first strand cDNA probe required for hybridization. Experiments 3 and 4 used Arcturus T7 RNA amplification to generate an aRNA target that was labeled with P33 using random 9mers. Experiment # 5 used 20 filters from the same lot of filters that had been hybridized and stripped on four previous occasions. Experiments #1-4 were done on Research Genetics GF211 filters lot 000223E - filters11, 12,13,15,16,17,18,19. Experiment #5 was done on lot 000223E - filters 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 69, 72, 73, 74, 75, 76, 77.
Exp 1 total RNA 9/20/01 |
|
|
UTA 1 |
# of Filters |
Phosphoimager Exposures |
|
10 million cpm per filter |
2 |
3 |
|
20 million cpm per filter |
2 |
3 |
|
40 million cpm per filter |
2 |
3 |
|
80 million cpm per filter |
2 |
3 |
|
|
|
|
Exp 2 total RNA 9/27/01 |
|
|
UTA 2 |
|
|
|
10 million cpm per filter |
2 |
3 |
|
20 million cpm per filter |
2 |
3 |
|
40 million cpm per filter |
2 |
3 |
|
48 million cpm per filter |
2 |
3 |
Exp 3 amplified total RNA 12/6/01 |
|
|
ARNA 1 |
|
|
|
1 ug starting RNA 170 million |
2 |
3 |
|
1 ug starting RNA 100million |
2 |
3 |
|
0.1 ug starting RNA 50 million |
2 |
3 |
|
0.1 ug starting RNA 25 million |
2 |
0* maximun intensity too low to import |
|
|
|
|
|
Exp 4 amplified
total RNA 12/20/01 |
|
|
ARNA 2 |
|
|
|
1 ug starting RNA 250 million |
2 |
4 |
|
1 ug starting RNA 250million |
2 |
4 |
|
0.1 ug starting RNA 250 million |
2 |
4 |
|
0.1 ug starting RNA 200 million |
2 |
4 |
|
|
|
|
Exp 5 total RNA 3/11/02 |
|
|
|
38 million cpm per filter |
21 |
2 |