October 17th, 2011
N59 - B. pseudomallei fimbriae
Progress Report 1
Progress, Results, and Discussion
The first round was a practice selection against lysozyme, using N34 RNA pool. This was accomplished under standard conditions – streptavidin beads, 1X PBS Selection Buffer (pH 7.4), 30 minute incubations at room temperature, three washes, one volume, 400:200 pmol ratio of protein: RNA. Following RNA immobilization on the beads, the pool binding reaction was denatured at 65C for five minutes, the protein was added, and the mixture was incubated per the above conditions This was washed again with 1X PBS, eluted with 200 ul of 80 C diH2O, and finally precipitated with ethanol (3 ul glycogen, 7 ul RNA, 77.3 mM NaOAc, and 1,110 ul 100% EtOH). The RNA underwent reverse transcription with the following steps: 1.5 ul diH2O, 20 mM N34 reverse primer, and 0.5 mM dNTPs for five minutes at 65 C, followed by cooling to 4 C; then 1X First Strand Buffer, 10 mM DTT, and 1 ul SS II reverse transcriptase were added, and the reverse transcription reaction was allowed to proceed over these steps: 42 C for 50 minutes, 70 C for 15 minutes, and slow cooling step to 4 C. Cycle course PCR was run according to the following mixture: 1X PCR buffer, 0.2 mM dNTP, 0.4 uM forward and reverse N34 primer, 2 ul RT DNA, 0.8 uL Taq DNA polymerase, and 78.2 ul sterile diH2O. During the cycle course PCR, 5 uL samples were taken from each wash at the conclusion of 6, 9, 12, 16, and 20 cycles.
Figure 1 – Cycle Course Lysozyme:
Legend - -------- = Wash #
-------- = Cycle #
-------- = 100 Base pair ladder
The 3.8% agarose gel indicates cycle 16 as slightly overamplified, so 14 cycle PCR was chosen for large scale. The W0 sample shows a slight amplification after 20 cycles, suggesting impurity of binders, but this amount appears to be fairly negligible.
As seen in Figure 1, the gel was successful, with very slight impurity in the W0; the large scale was run for 14 cycles, according to the same mixture and temperatures of cycle course. At this point, the B. pseudomallei fimbrial protein was delivered, and selection was restarted on the new target, with the following conditions: binding on streptavidin beads, N58 primer, 10X PBS Selection Buffer, 30 minute incubations at room temperature, 3 washes, 1 volume, 400:200 pmol ratio protein: RNA. This selection was run up to the cycle course step, according to the same protocol as the practice lysozyme round. It was then announced that the N58 pool might be contaminated, and was subsequently thrown out.
Selection was restarted using the N59 pool, with the same reaction and buffer conditions, and as demonstrated below in Figure 2, the cycle course gel was a success; 14 cycles appeared to be the optimal amplification period, and this was applied to the large scale PCR reaction.
Figure 2: Cycle Course Fimbrial Protein:
Legend - colors and labels are same as above.
The 3.8% agarose gel reveals a proper cycle number between 12 and 16. No impurities showed in the no-template negative control, labeled NTC. W0 shows a very slight band at 20 cycles, but as before, this appears to be a non-factor.
Transcription was run, according to the following protocol: 1X TNX Buffer, 10 mM DTT, 75 mM each of ATP, CTP, UTP, and GTP (in that order), 5 ul template dsDNA, 3 ul diH2O, and 2 ul of T7 enzyme solution at 42 C for three hours. A PAGE gel is scheduled next, after which Round 2 will commence.
The main problem was disposal of the N58 primers; this was overcome by restarting with a new pool to the lab (N59) that might not yet be contaminated. An additional problem was the impurity of the W0 in the practice round, but this appears to be relatively minor - with the same technique, the amount of this impurity was reduced in the fimbrial protein selection.
In addition to the bench work, a class solution of TE buffer was prepared and the box of primers was organized.
Conclusion and Future Work
A practice round of selection against lysozyme was run until arrival of the B. pseudomallei fimbrial protein. This was selected against with N58 until word of contamination, and was restarted with N59 pool. The current round is through the transcription step, with PAGE gel and RNA quantitation to come next.