5RACE 和 3RACE.docx

3RACE PCR 3 Rapid Amplification of cDNA Ends (RACE) PCR This technique is used to obtain the 3end of a cDNA, it requires some sequence information internal to the mRNA under study. The sequence information obtained from this technique can be utilised to obtain full length cDNA clones using the 5RACE technique. The method Ive used is loosely based on that given in PCR protocols: A guide to methods and applications, Academic Press Inc. The protocol given uses SuperScript II reverse transcriptase (Life Technologies) and Taq polymerase from Boehringer Mannheim. You will need to adjust the reagents according to the enzymes you use. Reagents: Sterile water (high purity, RNase-free) 5 x SuperScript RTase buffer (Life Technologies)100mM DTT2.5mM dNTPs RNase inhibitor (Pharmacia) SuperScript RTase (Life Technologies)10 x PCR buffer (Boehringer Mannheim, contains 15mM MgCl2 )500mM dNTPs Mineral oil RNA : Both total and polyA RNA are suitable for this technique. I recently compared the two and found the polyA RNA reaction to have the edge, but only just. I do recommend using polyA RNA for 5RACE though. Primers:- T17 Adapter primer (T17AP): GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT Adapter primer (AP): GACTCGAGTCGACATCG Gene specific primer: Designed from known sequence, ideally it should an 18-22-mer with an annealing temperature of around 56℃, where a G/C = 4℃ and an A/T = 2℃. cDNA 3’末端的快速扩增（3’-RACE） 反转录： 1、转移1pg至100ng的poly(A)+RNA或1ug总RNA到一只新的离心管。用水调整体积至9ul。将RNA样品在75℃变性5min，将离心管插入冰中冷却。 2、向这种含有已变性的RNA样品的离心管内一次加入如下试剂： 5×扩增缓冲液 10ul 20mmol/L4种d NTP混合液（ph8.0） 1.5ul 10umol/L（dT）17-接头引物（80pmol）? 8ul 约20单位/ul胎盘Rnase抑制剂 1ul 100-200单位/ul反转录酶 1ul H2O补足至 50ul 反应管温育在37℃反应60min，设置3支阴性对照管。在一支对照管中加入合成第一链c DNA反应所需要的所有试剂，但不加模板RNA；第2支对照管加入除了反转录酶外的所有试剂；第三支对照管加入除了引物外的所有试剂。这些对照管进行所有后续的实验步骤。设置这些对照管能保证cDNA产物不是由于污染或由RNA的自身引导所致。 3、用