Text from pdf for indexing 8-15-12
2015-01-13azim58 - Text from pdf for indexing 8-15-12
I would like to design primers to add a poly T slip site in front of the
inserted cDNA genes in the directional human
cDNA library so that all 3 reading frames of the insert are expressed.
This is necessary since translation will begin
from the prokaryote promoter rather than from any eukaryote promoter
present. Designing these primers is not
as straightforward as I thought initially because the 3' end of the
inserted genes will have a poly A tail. Therefore,
both the 3' end and the 5' end of the inserted gene will be "infused" to
the plasmid using oligos that contain a poly T
site. As I add the poly T site to the genes initially, I need to make
sure that both poly T oligos are not in the mixture
together at the beginning. Otherwise these oligos may "prime" in the
incorrect location.
Insertion Site of Plasmid
3' end plasmid sequence: CGGGGTACGATGAGA
5' end plasmid sequence: TTGATACCACTGCTT
In the kit, the 2nd strand synthesis is the last PCR that occurs before
the In-Fusion, and the In-Fusion reaction
does not make use of any oligos. Therefore, the last oligos the kit uses
are during this 2nd strand synthesis. The
kit uses the 5' PCR Primer II A and the 3' In-Fusion SMARTer PCR Primer
at this step. The sequence of the 5' PCR
Primer IIA is not given. The sequence of the 3' In-Fusion SMARTer PCR
Primer is given and is shown below.
3’ In-Fusion SMARTer PCR Primer (12 μM)
5’– CGGGGTACGATGAGACACCA-3’
This primer does not contain any poly Ts because these were already added
during a previous step.
I e-mailed Clontech to obtain the sequence of the 5' PCR Primer IIA, but
they would not give this sequence to me.
They said this was proprietary information that could not be disclosed.
Therefore, I won't be able to modify the
in-fusion primers to add the poly T when the cDNA genes are included into
the plasmid at first. I will have to do
the in-fusion exactly the way the company intended. Then once my genes
are already inserted into the plasmid, I
can insert the poly T site just before the 15 bp of homology on the
plasmid on the 5' end side of the genes as well
as mutate the 15 bp so that they no longer contain a stop codon in any of
the 3 reading frames. This can be
accomplished by linearizing the plasmid with the 15 bp after and the 15
bp preceding it. Then I can do an infusion
using primers that contain the 13 Ts along with the appropriate 15 bp of
homology. In the case of the 15
bp downstream of the 13 Ts I will introduce a point mutation to remove
any stop codons.
Linearizing the plasmid
At this stage, the genes will already have been inserted, and the plasmid
just needs to be linearized.
Insertion Site
Lin2
GGTGACGAACCCTAGGAGATCTC
5'
3'
Lin1 Order Sequence: TCGACCTGCAGGCATGCAAGC
Lin2 Order Sequence: CTCTAGAGGATCCCAAGCAGTGG
58.7 C OM Tm
TCGACCTGCAGGCATGCAAGC
58.1 C OM Tm
5' 3'
The plasmid will be linearized with these two Lin primers. An In-Fusion
reaction will then be performed using primers with 13 Ts
of overlapping sequence.
TCGACCTGCAGGCATGCAAGCTGCTTGGGATCCTCTAGAG TCGACCTGCAGGCATGCAAGC
Need to find stop codons in this sequence in the direction that the Lac
gene is transcribed.
GAGATCTCCTAGGGTTCGTCGAACGTACGGACGTCCAGCT
reverse
CTCTAGAGGATCCCAAGCAGCTTGCATGCCTGCAGGTCGA
complement
Stop Codons
TAG
TAA
TGA
The bottom strand specifies the codons and contains one stop codon. What
are all of the possible one base pair mutations I could
make from the codon TAG?
This strand specifies the codons
TAA
TAC
TAT
TCG
TTG
TGG
AAG
CAG
GAG
Stop
Tyrosine
Tyrosine
Serine
Leucine
Tryptophan
Lysine
Glutamine
Glutamic Acid
I think Leucine codon would be the most innocuous codon to mutate to.
Lin1
Performing In-Fusion
Insertion Site
TCGACCTGCAGGCATGCAAGCTGCTTGGGATCCTCTAGAG TCGACCTGCAGGCATGCAAGC
= codon to be mutated
Left Plasmid In-Fusion Site Right Plasmid In-Fusion Site
CTGCTTGGGATCCTCAAGAGAAAAAAAAAAAAA
58.1 OM Tm GACGAACCCTAGGAGTTCTC
5' 3'
58.1 OM Tm
TTTTTTTTTTTTTAGCTGGACGTCCGTACGTTCG
5'
3'
ATNL1 primer
ATNL2 primer
ATNL1 primer order sequence: CTGCTTGGGATCCTCAAGAGAAAAAAAAAAAAA
ATNL2 primer order sequence: GCTTGCATGCCTGCAGGTCGATTTTTTTTTTTTT
TCGACCTGCAGGCATGCAAGC