关键词: preparation fluorescent dna probe
来源: 互联网
1039 阅读
I. Preparing fluoresenctly labeled cDNA (probe):
Cy3 |
Cy5 |
||
mRNA (1 g/l ) |
x l |
y l | (2 m g of each if mRNA, 50-100m g if total RNA) |
Oligo-dT (4 g/l ) |
1 l |
1 l |
(Anchored: 5'-TTT TTT TTT TTT TTT TTT TTV N-3') |
ddH2 O (DEPC) |
to 15.4 l |
to 15.4 l |
|
Total volume: |
15.4 l |
15.4 l |
Reaction mixture |
l |
. . . . . |
Unlabeled dNTPs |
Vol. |
Final conc. |
5X first-strand buffer* |
6.0 |
dATP (100 mM) |
25 uL |
25 mM |
|
0.1M DTT |
3.0 |
dCTP (100 mM) |
25 uL |
25 mM |
|
Unlabeled dNTPs |
0.6 |
dGTP (100 mM) |
25 uL |
25 mM |
|
Cy3 or Cy5 (1 mM, Amersham) |
3.0 |
dTTP (100 mM) |
10 uL |
10 mM |
|
Superscript II (200 U/uL, Gibco BRL) |
2.0 |
ddH2O |
15 uL |
||
Total volume: |
14.6 l |
Total volume: |
100 uL |
* 5X first-strand buffer: 250 mM Tris-HCL (pH 8.3), 375mM KCl, 15mM MgCl2)
Select the appropriate row from the table:
Cover Slip Size (mm) | Total Hyb Volume (m l) | Probe & TE (m l) | 20x SSC (m l) | 10% SDS (m l) |
22 x 22 | 15 | 12 | 2.55 | 0.45 |
22 x 40 | 25 | 20 | 4.25 | 0.75 |
22 x 60 | 35 | 28 | 5.95 | 1.05 |
*20x SSC: 3.0 M NaCl, 300 mM NaCitrate (pH 7.0)
II. Washing and Scanning Arrays:
Wash | Description | Vol (ml) | SSC | SDS (10%) |
1A | 2x SSC, 0.03% SDS | 200 | 200 ml 2x | 0.6 ml |
1B | 2x SSC | 200 | 200 ml 2x | -- |
2 | 1x SSC | 200 | 200 ml 1x | -- |
3 | 0.2x SSC | 200 | 200 ml 0.2x | -- |
更有优质直播、研选好物、福利活动等你来!
本网站所有注明“来源:丁香园”的文字、图片和音视频资料,版权均属于丁香园所有,非经授权,任何媒体、网站或个人不得转载,授权转载时须注明“来源:丁香园”。本网所有转载文章系出于传递更多信息之目的,且明确注明来源和作者,不希望被转载的媒体或个人可与我们联系,我们将立即进行删除处理。
我的询价