SMARTer_RACE_5_3__Kit_User_Manual_022714(14)

clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual

4. Incubate tubes at 72°C for 3 minutes, then cool the tubes to 42°C for 2 minutes. After cooling, spin

the tubes briefly for 10 seconds at 14,000 x g to collect the contents at the bottom. NOTE: This step can be performed in a thermal cycler. While the tubes are incubating, you can

prepare the Master Mix in Step 6.

5. To just the 5’-RACE cDNA synthesis reaction(s), add 1 µl of the SMARTer II A Oligonucleotide

per reaction.

6. Prepare enough of the following Master Mix for all 5’- and 3’-RACE-Ready cDNA synthesis

reactions. Mix these reagents at room temperatures in the following order:

5.5 µl Buffer Mix from Step 1

0.5 µl RNase Inhibitor (40 U/µl)

2.0 µl SMARTScribe Reverse Transcriptase (100 U)

8.0 µl Total Volume

7. Add 8 µl of the Master Mix from Step 6 to the denatured RNA from Step 4 (3’-RACE cDNA) and

Step 5 (5’-RACE cDNA), for a total volume of 20 µl per cDNA synthesis reaction.

8. Mix the contents of the tubes by gently pipetting, and spin the tubes briefly to collect the contents at

the bottom.

9. Incubate the tubes at 42°C for 90 minutes in an air incubator or a hot-lid thermal cycler.

NOTE: Using a water bath for this incubation may reduce the volume of the reaction mixture (due to

evaporation), and therefore reduce the efficiency of first-strand cDNA synthesis.

10. Heat tubes at 70°C for 10 minutes.

11. Dilute the first-strand cDNA synthesis reaction product with Tricine-EDTA Buffer:

Add 10 µl if you started with <200 ng of total RNA.*

Add 90 µl if you started with >200 ng of total RNA.*

Add 240 µl if you started with poly A+ RNA.

*The copy number of your gene of interest should be the determining factor for diluting your sample.

If you have 200 ng of total RNA but your gene of interest has low abundance, dilute with 10 µl. If

you have 200 ng of total RNA and the gene of interest is highly abundant, dilute with 90 µl.

12. You now have 3’- and 5’-RACE-Ready cDNA samples. Samples can be stored at –20°C for up to

three months.

百度搜索“77cn”或“免费范文网”即可找到本站免费阅读全部范文。收藏本站方便下次阅读，免费范文网，提供经典小说综合文库SMARTer_RACE_5_3__Kit_User_Manual_022714(14)在线全文阅读。