October+3-7

October 3, 2011 Today we performed ligation. PCR is the creation of billions of copies of pur GFP DNA Ligation is the process of inserting our linear GFP DNA into the vector - we use restriction enzymes to cut the DNA at specific sites

Transformation- we heat shock the bacteria to get plasmid DNA inside - as soon as plasmid DNA is inside, the bacteria starts to transcribe from plasmid and make protein -> ampicillian and GFP protein - origin of replication: site on the plasmid where the bacteria's RNA polymerase binds and starts transcription - transcription and translation occur simultaneously in prokaryotes

October 5, 2011 1. Dump out the medium-> pellet in bottom 2. Add 250 microliters r3 buffer - this is used to suspend cells in 3. transfer 1 microlitrs tube 4. vortex for 30 seconds 5. Add 250 microliters L7 -> invert tube 10 times. - burst everything in cells open->plasmid-> lysis 6. Wait 5 minutes 7. Add 350 microliters M4 -> invert 10 times which will yield a percipitate 8. Centifuge for 10 minutes -> clear solution to column 9. centirfuge for 1 minute -> add 500microliters W10 10. cintriuge for 1 minute -> add 200 microliters of W10 11. Centrifuge for 1 minute W10 and W9 are wash buffers -> ethonal