SMARTer_RACE_5_3__Kit_User_Manual_022714(18)

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

6. Place a NucleoSpin Gel and PCR Clean-Up Column into a Collection Tube (2 ml) and load up to

700 µl of sample. Centrifuge for 30 seconds at 11,000 x g. Discard flow-through and place the

column back into the collection tube. Load remaining sample if necessary and repeat the

centrifugation step.

7. Add 700 µl Buffer NT3 to the column. Centrifuge for 30 seconds at 11,000 x g. Discard flow-through

and place the column back into the collection tube.

8. Centrifuge for 1 minute at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column

does not come in contact with the flow-through while removing it from the centrifuge and collection tube.

NOTE: Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of

ethanol can be achieved by incubating the columns for 2–5 minutes at 70°C prior to elution (Step 9).

9. Place the column into a new 1.5 ml microcentrifuge tube (not provided). Add 15–30 µl Buffer NE

and incubate at room temperature (18–25°C) for 1 minute. Centrifuge for 1 minute at 11,000 x g to elute DNA.

NOTE: DNA recovery of larger fragments (>1000 bp) can be increased by multiple elution steps

with fresh buffer, heating to 70°C, and incubation for 5 minutes.

B. Protocol: In-Fusion Cloning of RACE Products

For more details on the included In-Fusion HD Cloning Kit, please download its User Manual from our

website at .

1. Combine:

1 µl Lineareized pRACE vector (provided with SMARTer RACE 5’/3’ Kit Components)

7 µl Gel-purified RACE product (Section VII.A, Step 9)

2 µl In-Fusion HD Master Mix

10 µl Total Volume

2. Incubate for 15 minutes at 50°C and transfer to ice.

3. Follow the protocol provided with your Stellar Competent Cells to transform the cells with 2.5 µl of the In-Fusion reaction mixture.

IMPORTANT: DO NOT add more than 5 µl of the reaction to 50 µl of competent cells. More is not

better. Using too much of the reaction mixture inhibits the transformation.

4. Place 1/100–1/5 of each transformation reaction into separate tubes and bring the volume to 100 µl

with SOC medium. Spread each diluted transformation on a separate LB plate containing 100 µg/ml

of ampicillin.

5. Centrifuge the remainder of each transformation at 6,000 rpm for 5 minutes. Discard the supernatant

and resuspend each pellet in 100 µl fresh SOC medium. Spread each sample on a separate LB plate

containing the appropriate antibiotic. Incubate all of the plates overnight at 37°C.

6. The next day, pick individual isolated colonies from each experimental plate. Isolate plasmid DNA

using a standard method of your choice (e.g. miniprep). To determine the presence of your RACE

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