Modify+SfiI+and+NotI+062512

Now that I have my cut fragment (plasmid cut with SpeI and NotI), I can remove the SfiI, add a NotI, and remove the other NotI.

refer to Plan for modifying pComb3XSS vector 061312

Basically, I need to PCR my fragment with ANotI, and RNotI primers. Then I need to reamplify withANotI2, and RNotI2 primers. Then I need to perform the In-Fusion reaction with the plasmid backbone. This plasmid will need to be transformed into some bacteria. Then this plasmid will need to be sequenced with SANotI, and SRNotI.

I will use the Primestar Max DNA Polymerase

In-Fusion was transformed into E coli. Mara miniprepped this culture, and sent the plasmid for sequencing.

Item: MM In-Fusion DNA 6-29-12

Sequencing Results L:\storage\CIM Research Folder\DR\2012\7-5-12\sequence results

Conclusions from this analysis: The area around the forward primer was modified exactly as expected. The vector was modified as follows: The vector was cut with SpeI and NotI, and then this fragment was PCRd to remove the SfiI and add a NotI to this side as well as remove the NotI on the other side. The SfiI was removed, a NotI was added, and the polyT was added. The area around the reverse primer shows that the NotI was removed, but there is also some additional known sequence. This sequence does not affect anything of interest to me though.

Clustal_Input_0705121130 Clustal_Input_070512_0122 Clustal_Input_070512_1350

Alignment with whole plasmid 070512

7-9-12 Here is another set of sequencing results. I may actually not spend time looking at these, and just look at the sequencing results on the final plasmid. L:\storage\CIM Research Folder\DR\2012\7-9-12

7-19-12 Sent "In-Fusion 6-29-12 MM" plasmid for sequencing once again just to verify things. This was done since I had some sequencing problems when the second In-Fusion modification sequencing results did not look well.

Sequence Analysis L:\storage\CIM Research Folder\DR\2012\7-20-12\pcomb analysis

The SAPTF primer gave me an okay read, but it was too short. The SAPTR primer was in completely the wrong location. The SANotI and SRNotI primers worked well and as expected. When I resequence with some plasmid from a new miniprep, I will try multiple primer concentrations (1/2X, 1X, and 2X).