S-STEM Scholar Rhiannon Giesegh's Blog

 This week was finding  the equal molar for the heat gradient, the use of C1V1=C2V2. For left there was 33ng/μl and tet was 33ng/μl. Left needed to be 50ng and tet was 35ng. So the equation tuned out to be for tet (33ng/μl)(3μl)=x(35/6), and for left(33ng/μl)(3μl)=x(50/6). These were then used for a heat gradient. 

 During this week it was a testing week. This was a time to check the use of both our protocals, the ANNA protocal was the protocal was onethat there was the ability to see the DNA on a gel,while hldegarth was leaving streaks  in the gel, which was found out later. There was also a change of primer labeling. Now the left fragment of deinococus rediduresns, is with primers A,B. Tet fragment from E.coli labled with C,D, Right fragment from D.Rad with E,F.  We followed the protocals exactly to see if there was anyproblems with each protocal. The previous plasmid streaks on the gel were aqacounted for by this gel. We ran Left, Tet,and right fallowing the procedure exactly for both protocals.  This is the picture of the ANNA protocal From these gels there was the knowlage that there are streaks onthe Hilgarth protocal. So we decided to only go forward with the ANNA protocal. 

 This week was; testing our protocals. With a gel from last weekwas used to test dilution of primers. There was a mix up with  the protocals for hilgarth where step 3 was used insted of step 2 which is a change in touchdown protocal. Touchdown is a slow change of -0.5 per cycle, which is close to a heat gradient. Did dilutions for the hilgarth protocal of primers. We warmed a sample of both dilutedprimers and undiluted primers.  Also did a gel to gel clean up to runa gel, that still had streaks. Did a gel extraction, redid hilgarth step 2. These are the ANNA and Hilgarth programs,that show diluted and undiluted Diluted and undiluted primes Gel extraction gel From these gels show that there is still some genomic DNA carring over from some part of the process. This willneed to be checked to see if there is anything with the primers or the protocal. Had to use the nanodrop, the florometer was not working. 

 This week was done by using a different protocol. The protocols were then named the ANNA protocol and the HIlgarth protocol. The Hilgarth protocol includes a new procedure called a touchdown, which is when the PCR machine will slowly lowers the temperature of the reaction. This touchdown was done on different samples of our left fragment of deinococcus and the tetracycline fragment from E.coli. To do this two different samples went through the ANNA at 37*c and the Hilgarth at 72*c, then each of these samples was run through the step 3 final fusion of the Hilgarth protocol. The fragments of left and tet were not as successful as we thought they would be. As the left fragment reacted very well to the new touchdown protocol, tet did not. So the fragment of left and tet that was already created went through an ANNA protocol and another sample of left and tet went through the Hilgarth protocol. Step3 of the Hilgarth protocol needed diluted primers from 10x stock to 1x. So taking 1μl of...

 This is a continuation of the project so far. We are still taking the fragments from deinococcus radiodurans and  Escherichia coli   to create a linear plasmid. These fragments from D. Rad. should be 1000bp (base pairs), while the fragment from E. coli, should be 700bp. From there a PCR is conducted to amplify the fragments with a heat construction with primers. Following the amplification the overlap without primers is started with a left or right fragment from D.Rad and a tetronomycin gene and a promoter. Afterward adding the leftover fragment to the segment, this will finish our linear plasmid, which will then be place with in an existing plasmid to see growth of a bacteria.  This week was running gells to see if the plasmids in our tubes were indead the fragments we were supossed to have. We had some trouble with our fragments smearing on the gells we were running and to combate any impurites, the PCR water was tested as well as another gel cleanup kit was run. ...