Modify SfiI and NotI 062512

2015-01-13

azim58 - 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

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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

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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).

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