Week+of+October+24

__October 24th__ Today we ran through everything that we have done so far in order to establish what we will be doing from here.
 * We started out using Peyer plasmid DNA which is supposed to contain:
 * GFP
 * Ampicillin Resistance
 * AraC
 * Origin of Replication
 * With this plasmid, we did PCR. (We also did a run using old Peyer plasmid DNA)
 * Results: there were no definite results even though we used multiple Taqs (Green Mix, Peyer, Aprell's, and I Taq.
 * Then we transformed the old DNA into green colonies, but only the control worked.
 * Next, we did a mini-prep of the old DNA to get the plasmid out of the bacteria.
 * Because the transformations and ligations didn't work, we took the mini-preps of the old DNA and did the double digest to cut the GFP out. However, the restriction enzymes cut in too many places.
 * Set a plan for finishing the analysis of the old DNA:
 * Check the EcoR1 digest to see if the gel would show only one band as it is supposed to. The one band would mean that plasmid was only cut in one place by the enzyme and linearized.
 * Transform the mini-prepped DNA to see if we get green colonies
 * Then, because this plasmid is giving too much trouble we will be switching to using the pGlo plasmid. We will have the vector map and a map of cutting sites which would make it easier to prepare a stock for the GFP lab that is taken to the schools.
 * Using the pGlo, we will do a mini-prep, design forward and reverse primers for GFP PCR, and isolate large quantities of the the plasmid. We will then cut out the plasmid and linearize it.

__October 26th__ Today, we did a 5ml culture so that we can do a mini-prep to isolate the plasmid. When making the culture, we learned sterile technique over a flame to prevent contamination. We also ran a gel of the single digest of only EcoR1 from the 19th.