October 18, 2011
Fall 2011
Pool N34, HIV-1 Gag p24
Abstract and Proposal Link:
http://aptamerstream.blogspot.com/2011/08/aptamer-selection-against-hiv-1-gag-p24.html
Progress,
Results and Discussion:
In the week
following the completion of the second practice round, the RNA selection
process began against the HIV-1 Gag p24 protein. The conditions selected were
tailored for the protein itself, and will become more restricted in order to
select for the best binding aptamers. Because the protein was tagged with His,
nickel-NTA beads were used to incubate the RNA with the protein. This
incubation lasted for 25 minutes and was kept at 37*C for its duration, in
order to mimic body temperature. Three washes using one volume (100 uL) was
used to wash the loose or non-binding RNA from the beads, and the buffer used
was Tris at pH 7.3.
After
ethanol precipitation and reverse transcription were performed on the reaction,
cycle course was done on the resulting DNA. After withdrawing 5 uL from the E1,
W3, and W0 reactions every 6th, 9th, 12th, 15th,
and 20th cycle, ethidium bromide was added to each of the 15 tubes. The
reactions run for 35 minutes in TBE buffer at 110 V in a 3.8% agarose gel. It
was determined that the optimal cycle for running large scale PCR without
overamplifying the DNA was 10 cycles, as seen on Figure 1 below.
Figure 1: PCR gel for R1. The 3.8% agarose gel
was run at 110 V for about 35 minutes in TBE buffer. Samples were taken
starting at cycle 6 until cycle 15 in 3 cycle increments, then at cycle 20.
A no
template control was made for the cycle course PCR, however, it was not added
in with the cycle course samples due to human error. Therefore, it was run the
next day by itself in another 3.8% agarose gel for 30 minutes at 105 V in TBE
buffer. No bands appeared, therefore, all reagents were free of contaminants
(see Figure 2). Large scale PCR was also performed on 6 tubes of 100 uL of DNA
for 10 cycles. These reactions were then ethanol precipitated, and
transcription was performed on the result.
Figure
2: The no template control was run in a 3.8% agarose gel in TBE buffer at 105 V
for 30 minutes alongside the ladder. No bands appeared, and the small dot that
appeared was assumed to be caused by human error while taking the picture.
Problems
Encountered:
There were no
problems with working with the protein during target immobilization and
incubation, and binding and selection as well as ethanol precipitation and
reverse transcription went smoothly. However, when the cycle course gel was
run, it was noted that an unusual amount of bands appeared in W3. Although this
result is not unheard of, it is still undesirable as it shows that there are
many loose binders. In addition to this, during large scale PCR it was noticed
that instead of 600 uL of reagents, there were about 500 uL after PCR. This
change in volume could be due to evaporation or human error (i.e not adding a
reagent, which could cause failure while running the PAGE gel).
Additional Work:
When the lab used up all the 3.8% agarose gel, I helped Austin to prepare more, which was a great learning experience in case no mentors are present and the agarose is needed. I also helped him to aliquot 4 uM of dNTP’s when we ran out of stock.
Conclusion
and Future Work:
Since
research on HIV-1 Gag p24 has begun, most of a round of selection has been
completed. Cycle course was successful, even though there were more bands in W3
than desirable. The no template control was clear, and all that remains to
complete is the PAGE gel and elution of RNA. Over the next weeks I plan to
complete this round and about three or four more others, prior to the next
progress report.
P.S Gwen sorry for posting so late!!
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