Overlap-1

 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. The left+Tet fragment turned out to be better than expected but wose than previous gels. Following a right+Tet sample was perpared to see if that would produce a better result. There was a gel run on a heat grradient to see if there was a temperature problem. Following two different prrotocals started a left+Tet to connect a right fragment, the tube broke in the PCR machine during the process. 

Figure 1: A gel run to test a Gel cleanup kit and PCR cleanup 

Figure2: This was a confermation gel that Left+ Tet were infact together

Figure 4: This was a test of a uncleaned sample of left+tet, as well as a test for PCR water

Figure 5: These are a left and a Tet using a different PCR protocal

All of these gels show that while we have had a left+Tet fragment we have not had a right+Tet or a completed seegment. While the new protocal seems to work on the left and right fragments of D.Rad the Tet from E. coli does not enjoy the new protocal where in the old protocal it was one of the better band. During this it was discovered that the bacteria that we got the Tet from may have been contaminated and we are making more as a precaution. SO far in this experiment the uses of PCR techniques and the use of bacteria growth is used most often.