Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols/GelPurification

From OpenWetWare

Jump to: navigation, search

Image:DeoxyriboNucleicAwesomeHeader.jpg

Wednesday, July 23, 2014

Home

Members

Project

Protocols

Progress

Discussion

References



Gel Purification

1. Anneal the complex using a PCR machine

2. Wash the plates and dry for 45 minutes For 300ul sample, add 50ul BB_ND dye 170 μl of sample will be loaded per lane. Each sample will take 2 lanes, and one blank lane should be placed between different samples.

3. Make a 12% non-denaturing (ND) gel for purification Lulu Qian’s recipe for 40ml of 12% ND gel which works for one gel - 12ml 40% Acrylamide - 4ml 10x TAE/Mg++ - Fill with MQ water to 40ml total volume (add 24 ml) - 240ul 10% APS - 24ul TEMED

   Note: APS should be used within 10 days

4. Run the gel under 150V for roughly 4.5 hours ~ 6 hours (when the dye nearly runs off the gel) depending on the size of the sample. For example, if the length of DNA strand is 122nt~176nt, run for 6 hours. Change the buffer every 1.5 hours.


Personal tools