Week+of+October+3

__October 3rd__ Today we did an in-depth review of the theory involved in the cloning of the GFP.
 * 1) In ligation, we use the PCR products and insert the linear GFP into the vector in order to get a plasmid.

Linear PCR product being inserted into the circular vector. (picture from google images)

We also did our own ligations today that we will use on Wednesday to do mini preps.
 * 1) Restriction enzymes are used to cut the vector at specific sites in order to get the open vector that the DNA can attach to (in this case due to the sticky ends).
 * 2) The plasmid closes using the ligase.
 * 3) Transformation is when the bacteria is heatshocked in order to get the plasmid DNA inside the bacteria. (As soon as the plasmid is inside the bacteria, it starts to transcribe to make RNA and then protein that if it contains the GFP will be amicillin resistant).
 * The bacteria's polymerase bind and starts transcription at the point called the origin of replication. And becasue these cells are eukaryotic, the transcription and translation are done simultaneously.
 * 1) When the bacteria is plated and it begins to replicate, the plates contain ampicillin and arabinose. The ampicillin is to kill and bacteria that does not contain the ampicillin resistance that is in the plasmid, and the arabinose is to ensure replication will be completed.
 * The plasmid contains AraC operon. AraC operon, on its own, will cause the DNA to twist and it will prevent the replication of the GFP and cause it to be repressed.
 * With arabinose sugar, the plasmid will remain in the circle without twisting, and it will allow the polymerase to carry on with the replication, allowing the GFP to be copied.
 * 1) Therefore, when our bacteria is plated it can have the three following possibilities based what is on the plate:
 * Ampicillin- no GFP will show because the replication was repressed
 * Ampicillin and Arabinose- almost all GFP (some non-GFP could slip through)
 * Arabinose- mixture of GFP and non-GFP because there is no ampicillin to kill the unwanted bacteria.

__October 5th__ Today we used two colonies of our ligations to do did mini preps t o get a sample with a higher concentration of DNA that what we previously had. __Mini Prep "Recipe"__
 * 1) Dump out medium
 * 2) Add 250 milliliters of R3 (media to resuspend cells)
 * 3) Transfer to 1.5 milliliter tube
 * 4) Suspend pellet in the buffer
 * 5) Vortex for 30 sec
 * 6) Add 250 milliliters of L7 (bursts everything in the cells open)
 * 7) Invert the tube to make the solution syrupy
 * 8) Wait 5 mins
 * 9) Add 350 milliliters of N4 (neutralizes lysis reaction)
 * 10) Invert 10 times to get a percipitate
 * 11) Centrifuge for 10 mins
 * 12) Put clear solution in the column
 * 13) Centrifuge for 1 min
 * 14) Add 500 milliliters of W10 (washes the buffer)
 * 15) Centrifuge for 1 min
 * 16) Add 700 millilters of W9 ( washes the buffer)
 * 17) Centrifuge for 1 min (2x) [[image:http://biowww.net/techniques/var/ezflow_site/storage/images/media/images/plasmid-dna-mini-column/649-1-eng-US/plasmid-DNA-mini-column_imagelarge.jpg width="302" height="232" align="right" link="http://www.google.com/imgres?q=dna+mini+prep&um=1&hl=en&sa=N&qscrl=1&nord=1&rlz=1T4SKPT_enUS411US412&biw=943&bih=428&tbm=isch&tbnid=tS0HrydOWTjN9M:&imgrefurl=http://biowww.net/techniques/Media/Images/plasmid-DNA-mini-column&docid=4A6tOZf9MqzpZM&imgurl=http://biowww.net/techniques/var/ezflow_site/storage/images/media/images/plasmid-dna-mini-column/649-1-eng-US/plasmid-DNA-mini-column_imagelarge.jpg&w=302&h=232&ei=EUueToGEEKWKsALdv6DKCQ&zoom=1&iact=hc&vpx=580&vpy=123&dur=2770&hovh=185&hovw=241&tx=161&ty=130&sig=113980810863664117756&page=1&tbnh=77&tbnw=109&start=0&ndsp=15&ved=1t:429,r:13,s:0"]]

This is a photo of what is needed to make a miniprep. The larger tubes are eventually what the DNA will be in. The smaller cylinder looking containers are the columns.

(photo from google images)