SMARTer_RACE_5_3__Kit_User_Manual_022714(9)

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

IV. Primer Design

A. Primer Sequence

Gene-Specific Primers (GSPs) should:

be 23–28 nt to ensure specific annealing

be 50–70% GC

have a Tm ≥65°C; best results are obtained if Tm >70°C, which enables the use of touchdown PCR.

(Tm should be calculated based upon the 3’ (gene-specific) end of the primer, NOT the entire primer.)

not be complementary to the 3’-end of the Universal Primer Mix

Long primer = 5’–TAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT–3'

Short primer = 5’–CTAATACGACTCACTATAGGGC–3’

be specific to your gene of interest

both have 15 bp overlaps with the vector at their 5’ ends (i.e., add the sequence

GATTACGCCAAGCTT to the 5’ ends of both GSPs’ sequences; see details below)

The relationship of the primers used in the SMARTer RACE reactions to the template and resulting

RACE products are shown in detail in Figure 3.

For the complete SMARTer RACE protocol, you will need at least two GSPs: an antisense primer for the

5’-RACE PCR and a sense primer for the 3’-RACE PCR. If you are performing only 5’- or 3’-RACE,

you will only need one GSP. In our experience, longer GSPs with annealing temperatures above 70°C

give more robust amplification in RACE, particularly from difficult samples; however, there is generally

no advantage to using primers with gene-specific sequence longer than 30 nt.

Successful In-Fusion cloning requires a 15 bp overlap with the linearized vector. Given this, you will need

to add the sequence GATTACGCCAAGCTT to the 5’-end of your 5’ and 3’ GSPs to facilitate In-Fusion

cloning of your RACE PCR products. This specific sequence is in addition to the 22 nt gene-specific

sequence described above. The provided linearized pRACE vector already contains this overlap with the

Universal Primer A Mix included for PCR, and adding this sequence to the 5’-end of your GSPs will

complete the necessary overlap for the cloning reaction. Please note that the In-Fusion User Manual

contains only general primer recommendations that should not be used for this particular protocol.

Figure 3. The relationship of gene-specific primers to the cDNA template. This diagram shows a generalized first-strand

cDNA template. This RNA/DNA hybrid does not precisely represent either the 5’- or 3’-RACE-Ready cDNAs. For a detailed

look at those structures, see Appendices B & C. Note that the gene-specific primers designed here contain tails with In-Fusion

homology, and also produce overlapping RACE products. This overlap permits the use of the primers together in a control PCR

reaction. Additionally, if a suitable restriction site is located within this region, it will be possible to construct the full-length

cDNA by subcloning.

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