SMARTer_RACE_5_3__Kit_User_Manual_022714(15)

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

VI. Rapid Amplification of cDNA Ends (RACE)

PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING.

At this point, you have 3’- and 5’-RACE-Ready cDNA samples. The RACE reactions in this section use only a fraction of this material for each RNA of interest. There is sufficient single-stranded cDNA for PCR amplification of multiple genes.

A. Things You Should Know Before Starting RACE PCR Reactions

If you intend to use LD PCR to construct your full-length cDNA after completing 5’- and 3’-RACE, be

sure to set aside an aliquot of the 5’-RACE-Ready cDNA to use as a template in the PCR reaction.

Please note that the efficiency of RACE PCR depends on the abundance of the mRNA of interest in your

RNA sample. Additionally, different primers will have different optimal annealing/extension

temperatures. Refer to the Troubleshooting Guide (Appendix A) for suggestions on optimizing PCR conditions.

NOTE: This is a RACE-specific protocol. It differs from the general SeqAmp protocol in many regards.

B. Protocol: Rapid Amplification of cDNA Ends (RACE)

This procedure describes the 5’-RACE and 3’-RACE PCR reactions that generate the 5’ and 3’ cDNA

fragments. We recommend that you also perform positive control 5’- and 3’-RACE using the TFR

primers and UPM. Although the Universal Primer Short (UPM short) is provided, nested PCR is

generally not necessary in SMARTer RACE reactions.

1. Prepare enough PCR Master Mix for all of the PCR reactions plus one extra reaction to ensure

sufficient volume. The same Master Mix can be used for both 5’- and 3’-RACE reactions. For each

50 µl PCR reaction, mix the following reagents:

15.5 µl PCR-Grade H2O

25.0 µl 2X SeqAmp Buffer

1.0 µl SeqAmp DNA Polymerase

41.5 µl Total Volume

2. Prepare PCR reactions as shown below in Table 2. Add the components to 0.5 ml PCR tubes in the

order shown and mix gently.

Table 2. Setting up 5'- and 3'-RACE PCR Reactions

Component

5’- or 3’-RACE-Ready cDNA

(experimental)

10X UPM

5’ or 3’ GSP (10 µM)

H2O

Master Mix (Step 1)

Total Volume 5’- or 3’-RACE Sample 2.5 µl 5 µl 1 µl — 41.5 µl 50 µl UPM only (– control) 2.5 µl 5 µl — 1 µl 41.5 µl 50 µl GSP only (– control) 2.5 µl — 1 µl 5 µl 41.5 µl 50 µl

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(15)在线全文阅读。