Vincent Parker Notebook

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Revision as of 21:20, 26 June 2007

Vparker 17:11, 20 June 2007 (EDT) The sequences of the oligos I designed are as follows:

   VP1AF-cgtaaAGATCTATGAGTCGTTTAGTCGTAGTATC
   VP2AIF-CGATGACATTATCTGGATtCACGATTATCACCTG
   VP3AIR-CAGGTGATAATCGTGaATCCAGATAATGTCATCG
   VP4AR-cgtatCTCGAGttaGGATCCTTACGCAAGCTTTGGAAAGGTAGC
   VP5BF-cgtaaAGATCTATGACAGAACCGTTAACCGA
   VP6BIF-GCAACGTATTACTCAGATtTGGCCACAAATGGCG
   VP7BIR-CGCCATTTGTGGCCAaATCTGAGTAATACGTTGC
   VP8BR-cgtatCTCGAGttaGGATCCTTAGATACTACGACTAAACG

The oligos were named according to my initials, numerical sequence, and letters determining direction of oligo. Ex. VP1AF- Vincent Parker 1 otsA Forward.

Vparker 15:04, 21 June 2007 (EDT) After a long wait the oligos that I have ordered have arrived. I ordered 8 which PCR the two genes and get rid of the internal restriction sites. The template used to make the oligos was called MG 1655. In the ApE file which was split into two genes they were called ostA and otsB. They came in powder form and we have to dilute them down to 10 micro mols. That makes my master supply of my oligos when i use them i have to dilute them down to 100 micro mols. Using my oligos I can determine my PCR products... otsA will be broken up into two parts; PartA is 413 bps and PartB is 1077 bps. OtsB; PartA 485bps, PartB 381bps. I will PCR them overnight and digest tomorrow. Part A of otsA will be called V1-3 because i am using oligos 1 and 3 to PCr that specific part. Part B of otsA will be called V2-4. Part A of otsB will be called V5-7 and part B of otsB will be called V6-8.

VParker 16:37, 22 June 2007 (EDT) Big day planned. I have to take out my tubes V1-3, V2-4, V5-7, and V6-8 from the PCR machine.These are the 4 parts to my two genes. V1-3 is Part A of my otsA gene, it ends at the internal restriction BamHI site. V2-4 is part B to my otsA gene and it starts with the BamHI restriction site. I have already designed my oligos which code for a silent mutation within the BamHI site and codes for Leucine. V5-7 is part A of my otsB gene and ends at an internal BglII restriction site. V6-8 is part B of my otsB gene and starts at the BaglII restriction site and I have designed the oligos for that gene to have a silent mutation in the BglII site that codes for Isoleucine. As i came in to PCR my two genes, otsA and otsB, with the 8 oligos i designed. Two oligos per part. The oligos are named by number so when I mention V1-3 i am referring to part A of otsA but it will be PCRed with the first and 3rd oligos. Unfortunately when i came into the lab it was flooded and all I managed to do was take out Hannah's tube and my four out of the PCR machine and put them into the freezer for Monday. I did manage however to go home and sleep some more :-)

Vparker 16:02, 25 June 2007 (EDT) Today i came into the lab and found that 3 out of the 4 PCR tubes were in the freezer but that i have lost the tube marked V6-8. I had the PCR products from the tubes V1-3, V2-4, and V5-7. After having fumed a bit over my lost PCR I went to work on setting up a new PCR tube for V6-8 and to also clean tubes V1-3, V2-4 and V5-7. Austin was kind enough to share the PCR machine with me and i put in the tube V6-8 in and also the sewing reaction i was working on named V1-4. V1-4 is a combination between V1-3 and V2-4. Since V1-3 and V2-4 are two parts of one gene and I had to PCR out the internal restriction site. File:Firstgel.jpg Decided to try my hand in Agar plates today so I volunteered the High School team to learn how to prepare and pour them since Arthur was not here. They turned out looking good with no bubbles. So i was behind today in terms of my PCR and i have to digest and ligate tomorrow instead of today. Losing the V6-8 tube set me back an hour and a half so i will save the rest for tomorrow.

Vparker 15:04, 26 June 2007 (EDT) Started off today with digestion of tubes V1-4 and V5-8 which are my genes that i PCRed. V1-4 was my otsA gene and V5-8 was my otsB gene. atfer storing them in cultivation for an hour and a half Sam showed us how to prepare Agar solution and broth. we just put them in the autoclave machine and are waiting for both digestion and the autoclave to finish.