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From 2010.igem.org

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Revision as of 11:20, 21 October 2010

PROTOCOLS

 

Preparing 1% Agarose Gels:

Added 180 mL 1x TBE and 1.8g agarose

Heated up until agarose was no longer visible

Let cool (approx. 10 min)

Added 180 սL Ethidium bromide (1000X)

 

PCR Purification

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 50  µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

 

Heat shock transformation of the plasmids into our bacteria

1. Left cells and ligation reaction products on ice.

2. Added plasmid (5սL) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).

3. Left cells on ice for 30 min.

4. Applied heat shock of 45 seconds in 42C bath.

5. Put tubes on ice for 2 min.

6. Added 250 սL of LB (room temp.)

7. Incubated 1 hour at 37 C

8. Plated 100սL and left plate in the 37 degrees incubator  

9. Incubated overnight at 37C.