Week+November+7

__November 7th__ Today we did a double digest with Nhe1 and Sma1 to cut the plasmid into its GFP and vector components. This will allow us to later cut out the vector and GFP bands so that we can build up stocks of each. We also did a culture of our DNA so that we will have a larger supply.

__November 9th__ We ran a gel of Aprell's redo of our double digest. (Our original double digest did isolate the GFP like we wanted but the vector was cut into four segments. So, Aprell redid the digest using Sma1, and then Nhe1 seperate. We found that the original digest did not work because the loading buffers needed for each of the restriction enzymes was interacting in a bad way.) When running this gel, we used .8 agarose for a lighter gel when using the columns to later extract the GFP by dissolving the gel. We also had to cover the gel with foil in order to minimize the amount of light that interacts with the plasmid. We also did two more cultures of DNA. Then we learned how to plate bacteria and make our own spreaders. Plating Bacteria (from Google Images)

For homework, Paige and I designed primers for different restriction enzymes. When designing primers, you need a forward and reverse primer that will amplify the GFP sequence and contain the appropriate restriction sites. The primer needs to be about 17 to 20 nucleotides long and contain 45%-55% G's and C's. ( This is because G's and C's have triple bonds and will need a higher annealing temperature. Too many or too few will cause the restriction and primers to be thrown off. __Primers % G's and C's__ Forward Nhe1:
 * 5'_GCTAGCAAAGGAGAAGAACT_3' 9/20= 45%**

Reverse Sma1: Original ssDNA: 3'_GGGCCCATGGCTCGAGCTTA_5' 5_'CCCGGGTACCGAGCTCGAAT_'3 13/20= 65% 5'_CCCGGGTACCGAGCT_3' 11/15= 73% Forward HindIII: 5'_AAGCTTACCCTTAAATTTAT_3' 5/20= 25% 5'_AAGCTTACCCTTAAATT_3' 5/17= 29% 5'_AAGCTTACCCTTAA_3' 5/15= 33% 5'_AAGCTTACCCTTAAATTTATTTGGA_3' 7/25= 28% 5'_AAGCTTACCCTTAAATTTATTTGG_3' 7/24= 29%
 * 5'_CCCGGGTACCGAGCTCGAATT_3' 13/21= 61%**

Reverse HindIII: Original ssDNA: 3'_TTCGAACGTACGGACGTCCA_5' 5'_AAGCTTGCATGCCTGCAGGT_3' 11/20= 55% 5'_AAGCTTGCATGCCTGCA_3' 9/17= 52.3%

(The bolded primers are those that we decided will work best from our plasmid.)