Phase  

PCR of 16S-23S rRNA for Cloning

Mark Brown, Ian Hewson, Mike Schwalbach, Alison Davis - 2004

Details  

• Dilute your target DNA to 2.5 ng/ul in DIW.

• Make the master mix:                       

^Top

With unamended negative control.

• Use the following cycling conditions:

Hot Start 3 min at 94 deg C

------------------------------

Denature 94 deg C for 30s

Anneal 55 deg C for 30 s          x 30 cycles                  

Extend 72 deg C for 90 s

-------------------------------

with a final extension step of 72 deg C for 7 min

4 deg C chill at end of cycle    

• After PCR is finished, run a gel in 1 X TBE to ensure product formation and negative control(s). Load 5 ul of the PCR reaction into each well + 5 μl loading dye mixed in the PCR tube. Run gel at 100 V for 1 h (depending on gel length). Stain gel for 15 min with 20 ul / ml SYBR Gold.

• Purify PCR products using Zymo Clean & Concentrator -5 gel extraction kit (see kit protocol).

• Quantify DNA after elution using PICO green kit (see separate protocol).

• If low or no DNA, do not proceed.