Team:UTP-Panama/After Regional Week 2

Octuber 24 to 28

October 24, 2011

Morning
This day there was made an electrophoresis of digestion products which became on September 22, 2011 (the upstream and downstream). Following the protocol for electrophoresis:
1. Preparation of 1% agarose.
2. Moisten the chamber walls.
3. In a 70 mL Erlenmeyer flask add the agar and put a minute in the microwave, then take it off shake it and put it in the microwave 20 seconds.
4. Ethidium bromide placed in the microwave and cooled with tap water to a temperature between 60 ° C and 70 º C. 5. Placed between 2.5 to 5 micolitros Ethidium bromide. We mix before pouring it into the camera.
6. Once the gel solidified cover it with TAE (1X). (A)
7. 35μL were added ultra-filtered water plasmids were 65.5 ° C.
8. Droplets were eliminated on the walls with the sping-down applied to the tubes.
9. We put the tubes in the microwave 5 minutes.
10. Add loading buffer which was frozen thawed. How?
• Put dots of paper loading buffer.
• Of the 35 uL of plasmid take 10 mL (B).
• Re mix in the dots and then the wells.
11. A and B meet. Plasmid preparation and gel electrophoresis.
12. The plasmids with the loading buffer in the wells
Note: It is important to place the (-) side of the DNA
13. We ran the gel for 2 hours.
Our Upstream is BBa_K410000 and the Downstream is BBa_K381001

Afternoon At this time a purification protocol was required but in the gel electrophoresis previously made was not enough genetic material to recover so do not purified at this time.
To remember our goal with these protocols a flow chart was made and is presented below.

October 25, 2011

Purification Protocol: Extraction of 40 bp to 50 kb DNA fragments from Agarose Gels

Information necessary for the purification: BBa_K381001: 986bp BBa_K410000: 1517bp