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.

__GGCCCAGCCGGCC__ATGGCCCAGGTGCAGCTGCAGCAGTCAGGACCTGAGGTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGAAACACATTCACTGACTATATTGTAAGCTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAGATTTATCCTGGAAGCGGTACTACTTACTACAATGAAAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCCCTCTACTATGATGATGCTATGGACTACTG__GGGCCAAACGGGCC__CGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCGAGCTCACTCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCGTGACCTGCAGTGCCAGCTCAAGTATTAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAACTCTGGATTTATAGCACATCCAACTTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGTGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTACAGTGGTTACCACTCACGTTCGGCTCGGGCACAAGCTGGNAATCAAACGGGCGGCCGC

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) CAGCTGCAGCAGTCAGGACCTGAGGTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGAAACACATTCACTGACTATATTGTAAGCTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAGATTTATCCTGGAAGCGGTACTACTTACTACAATGAAAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCCCTCTACTATGATGATGCTATGGACTACTG__GGGCCAAACGGGCC__CGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCGAGCTCACTCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCGTGACCTGCAGTGCCAGCTCAAGTATTAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAACTCTGGATTTATAGCACATCCAACTTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGTGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTACAGTGGTTACCACTCACGTTCGGCTCGGGCACA

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