abstract
Proposal
Target: SATB-1
The Target had to be functionalized with biotin. The molecular weight of the protein was about 1,718 daltons; this lower than the minimum weight of most proteins that are biotinylated. I chose to biotinylate 9,000 pmol of the protein. For the desalting procedure my first desalt had 30 ul of sample while the 2nd desalt had 45 ul of sample. When all was said and done I recovered about 50% of my biotinylated target but since my target is so small the percentage did not call for concern. Since the target is functionalized with biotin, the bead used during selection was streptavidin.
Round One Conditions:
Target: SATB-1
Beads: Streptavidin
Pool: N-34
Incubation: 25 minutes at 25 degrees Celsius
Buffer: 10X HEPES
Wash Volume and Number: 3 washes and 2 volumes
Ratio of pool to target (pmol): 200:61
Progress Results and discussion:
Round one was very successful and round two though successful as well, there were problems encountered at the end of it. I entered round one with 61 pmol of the biotinylated target and 200 pmol of the N-34 pool. The 1X selection buffer had been pre-made so I proceeded to immobilize the target on to the streptavidin beads. A binding reaction was created using the HEPES buffer allowing an incubation of the pool of the target to take place. During binding and selection three washes were conducted. The RNA was eluted followed by a successful ethanol precipitation. The RNA turned into DNA by means of reverse transcription. The subsequent cycle course showed a similarity in the amplification of the wash three and the elution. This led me to conclude that more washes had to be done in the next round and that potentially the next round should be a negative selection. After consulting with Gwen I went ahead and performed a large scale pcr using cycle 14 of the cycle course. An ethanol precipitation of the large scale and an overnight transcription followed thereafter. The PAGE gel was successful on the first try and the nanospec showed that 90.6 ng/uL was recovered.
Figure 1: Cycle Course PCR gel for round 1
Round 2 conditions:
Target: SATB-1
Beads: Streptavidin
Pool: N-34
Incubation: 25 minutes at 25 degrees Celsius
Buffer: 10X HEPES
Wash Volume and Number: 4 washes and 2 volumes
Ratio of pool to target (pmol): 200:61
Round two was a negative selection meaning that I incubated two separate bead mixtures before binding and selection. The round was conducted with an initial concentration of the RNA from round one. This concentration was 90.6 ng/uL. The same pool to target ratio was used in this round. One sample consisted of the pool and streptavidin beads and the other of the target and streptavidin beads. During binding and selection four washes with two volumes were conducted to the results from the previous round’s cycle course. The RNA was then eluted and an ethanol precipitation was conducted. Reverse transcription was once again done to convert the RNA to DNA. The cycle course from this round showed good results in that the eluted species showed up better than the last wash. Doing an extra wash solved the problem seen in the first cycle course gel. The large scale pcr was done for cycle 15 of the cycle course. An ethanol precipitation of the large scale followed along with the performing of an overnight transcription. A successful PAGE gel was finally done but there was no time to nanospec.
Figure 2: Cycle Course PCR gel for round 2
Problems:
The only problem that was seen in round one presented itself in the results from the cycle course gel. The amplification of the wash three was similar to that of the eluted species. This was corrected by doing a negative selection in round 2 in addition to performing an extra wash. Round 2 proved to be the most troublesome. I accidently discarded the supernatant for wash 0 so the round had to be done without a wash 0. Reverse transcription had to be redone due to the fact that the power in lab went off while the sample was in the thermo cycler. The first PAGE gel ran did not work due to the leaking of the rig and the lack of current need to run the sample. The second PAGE gel was ran at 316V meaning that it was to take about two hours to complete but after and hour and 45 minutes the sample ran into the water. The third and fourth PAGE gels that were poured did not polymerize correctly. The fifth PAGE gel showed three partial shadows in three different places and this was due to the fact that when removing the gel from the plates the gel broke apart into little pieces rendering the gel useless. A sixth PAGE gel was done which showed good results.
Conclusions and Final work:
Round one and two showed good results with the concentration in round one being 90.6 ng/uL. In the future more subsequent rounds will be performed.
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