Oct.+2011

toc

**Week of the 3**
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
 * October 3, 2011 **

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

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
 * October 5, 2011**

Week of the 10
Today we nano-dropped our DNA from last week and the test results were good. We tested our two colonies we made into 4 tubes. We made more PCR using April's taq for one and the green mix for the other, 1 used my DNA colony 1A for both PCRs
 * October 10, 2011**

For Aprils Taq For the Green Mix
 * ~ **Reagent** ||~ **Amount** ||
 * 5x PCR Buffer || 4 microliters ||
 * DNTP || 5 microliters ||
 * GFP For. || 3 microliters ||
 * GFP Rev. || 3 microliters ||
 * DNA Template || 1 microliter ||
 * Taq Polymerase || 1 microliter ||
 * Water || 3 microliters ||
 * < **Total** ||< **20 microliters** ||
 * ~ Reagent ||~ Amount ||
 * Green Mix || 10 microliters ||
 * GFP For. || 3 microliters ||
 * GFP Rev. || 3 microliters ||
 * DNA Template || 1 microliter ||
 * Water || 3 microliters ||
 * **Total** || **20 microliters** ||

We prepared another gel to test our PCR product using our DNA made from the previous week. From the gel we could tell that both Colony 1A and 1B were succesful using the green mix and April's Taq.
 * October 12, 2011**


 * ~ Reagent ||~ Amount ||
 * Buffer || 2 microliters ||
 * Plasmid || 5 microliters ||
 * Agel || 1 microliter ||
 * ECPRl || 1 microliter ||
 * BSA || 2 microliters ||
 * Water || 9 microliters ||
 * **Total** || **20 microliters** ||

Week of the 17
Today we ran a double digest and by looking at the gel we ran afterward we discovered that it had failed. We are now looking to see why it cut in too many places on our vector.
 * October 17, 2011**


 * October 19, 2011**

Today we did a Ecor1 digest and used and made the master mix for it including the following:

(microliters) ||
 * ~ 6x(microliters) ||~ Reagent ||~ 1x
 * 12 || React 3 Buffer ||  ||
 * 12 || 10x BSA ||  ||
 * .3 || Enzyme ||  ||
 * 83.7 || Water (H2O) ||  ||
 * || DNA || 2 ||

We then used 18 microliters of the master mix and 2 microliters of DNA for each tube.

Week of the 24
**October 24, 2011** Today we took notes on our objectives as shown below:

** October 26, 2011 ** We ran a gel using our single digest from last week and the results turned out as followed: - ampicilian - broth for cells - coloney of GFP plasmid
 * We discovered that the Ecor1 didn't cut so we must find another primer to use.
 * Mini-culture:
 * the mini-culture isolated of DNA GFP plasmid for our mini-prep