More information about the insert that Barbara Lipes put in

2015-01-13

azim58 - More information about the insert that Barbara Lipes put in

Here is an e-mail from Barbara Lipes on October 12th.

Hi Kurt,

Here is the original info I sent to Tien regarding this construct:

Enclosed is a pellet of ethanol precipitated plasmid. The pellet should
contain at least 10 micrograms of DNA. The construct consists of pCANTAB
5E with an scFv insert (clone 1D9). The VH sequence just before the
linker region has been modified to contain an additional Sfi I site to
improve efficiency of Sfi I digestion. The insert sequence of 1D9+Sfi I,
from the 5' Sfi I site through Not I site is shown below with the Sfi I
sites underlined. To prepare vector for library cloning, perform
sequential digests with Not I and Sfi I.

GGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGCAGTCAGGACCTGAGGTGGTGAAGCCTGGGGCTTCAGTGAA
GATGTCCTGCAAGGCTTCTGGAAACACATTCACTGACTATATTGTAAGCTGGGTGAAGCAGAGAACTGGACAGGG
CCTTGAGTGGATTGGAGAGATTTATCCTGGAAGCGGTACTACTTACTACAATGAAAAGTTCAAGGGCAAGGCCAC
ACTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTA
TTTCTGTGCCCTCTACTATGATGATGCTATGGACTACTGGGGCCAAACGGGCCCGGTCACCGTCTCCTCAGGTGG
AGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCGAGCTCACTCAGTCTCCAGCAATCATGTC
TGCATCTCCAGGGGAGAAGGTCACCGTGACCTGCAGTGCCAGCTCAAGTATTAGTTACATGCACTGGTACCAGCA
GAAGTCAGGCACCTCCCCCAAACTCTGGATTTATAGCACATCCAACTTGGCTTCTGGAGTCCCTGCTCGCTTCAG
TGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGTGTGGAGGCTGAAGATGCTGCCACTTATTACTG
CCAGCAGTACAGTGGTTACCACTCACGTTCGGCTCGGGCACAAGCTGGNAATCAAACGGGCGGCCGC

===========================================================================
Ordering primers to sequence plasmid from this insert put in from Barbara
Lipes

Used Primer3 to design primers

Sequence used to input into primer3 (I cut off about 25 bp from the
original insert sequence)
CAGCTGCAGCAGTCAGGACCTGAGGTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGAAAC
ACATTCACTGACTATATTGTAAGCTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAGATTTAT
CCTGGAAGCGGTACTACTTACTACAATGAAAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAAC
ACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCCCTCTACTATGATGAT
GCTATGGACTACTGGGGCCAAACGGGCCCGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCT
GGCGGTGGCGGATCGGACATCGAGCTCACTCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACC
GTGACCTGCAGTGCCAGCTCAAGTATTAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAACTC
TGGATTTATAGCACATCCAACTTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTAC
TCTCTCACAATCAGCAGTGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTACAGTGGTTACCACTCA
CGTTCGGCTCGGGCACA

Output from primer3

These primers would amplify this insert, but I actually want to amplify
the part of the region outside of this insert so I will take the reverse
complement.

OLIGO start len tm gc% any 3' seq
LEFT PRIMER 96 20 60.13 50.00 4.00 0.00 AAGCTGGGTGAAGCAGAGAA
RIGHT PRIMER 684 20 60.18 55.00 4.00 3.00 AGCCGAACGTGAGTGGTAAC
SEQUENCE SIZE: 692 INCLUDED REGION SIZE: 692

Reverse Complements that I need
Left primer: TTCTCTGCTTCACCCAGCTT
Right primer: GTTACCACTCACGTTCGGCT

Now we can look at the alignment of these primers with the original
insert.

Form used to order

Invitrogen_ Oligo Order 10-21-11 KW.xls
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-19-2014d1132\01-19-2014d1338\Invitrogen_ Oligo Order 10-21-11 KW.xls"

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