Difference between revisions of "5' RACE"

Revision as of 14:36, 4 December 2012

Materials

• 5' RACE Kit (Version 2.0 from Invitrogen; http://products.invitrogen.com/ivgn/product/18374058)
• Double distilled water
• Absolute ethanol
• Total RNA, 1-5 ug
• Gene Specific Primers (see Designing Primers for 5' RACE )

Protocol

Copied from the manufacturer's protcol here

Preparation

• Resuspend gene specific primers to 1 uM
• Prepare and chill wash buffer by adding 18 mL water, 21 mL ethanol and 1 mL wash buffer concentrate into a glass bottle. Place at 4C
• Prepare and chill 70% ethanol by adding 35 mL ethanol to 15 mL water in a glass bottle. Place at 4C

First Strand Synthesis

1. Combine the following in a PCR tube:

Component	Amount
GSP1	2.5 pmoles (2.5 uL of 1 uM)
RNA	1-5 ug (from nanodrop)
DEPC Water	To 15.5 uL

2. Incubate at 70C for 10 min to denature RNA using the PCR Program 5' RACE 70C
3. Place on ice for 1 min
4. Add the following in order:
1. 2.5 uL 10X PCR Buffer
2. 2.5 uL 25mM MgCl2
3. 1 uL 10 mM dNTP
4. 2.5 uL DTT
5. Incubate 1 min at 42C using the PCR program 5' RACE First Strand, which covers the next three steps
6. Add 1 uL SuperScript II RT
7. Incubate 50 min at 42C
8. Incubate 15min at 70C
9. Remove and add 1uL RNAse H, mix thoroughly
10. Incubate 30min at 37c using the PCR Program 5' RACE RNAse H. Can freeze at -20 or continue to cDNA Purification

cDNA Purification