IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-18

From OpenWetWare
Jump to: navigation, search

Logistics Communications

Streptavidin Magnetic Beads, Take 3-4

  • Trypsinized the beads, in 50uL solution that were retained from yesterday, for four hours @ 37[[:Category:{{{1}}}|{{{1}}}]].
  • Ran gel of them, ladder, and p7308
    • Noticed when took the gel out for imaging that the buffer was below the gel slightly - didn't cover top
    • Imaging: NO BANDS AT ALL, not for the ladder or even p7308.
      • Suspect that the gel didn't run due to too-low-buffer.
  • On the off-chance that the trypsin digest didn't work, used 2uL (of unknown activity concentration, see above) of proteinase K to try again to digest the streptavidin.
    • Incubated overnight at 37[[:Category:{{{1}}}|{{{1}}}]].
  • Mixed c5.0.1 and other pre-working stocks and working stocks listed above.
  • Folded 4 rxns each, at intermediate {Mgcl2}} of 20mM final concentration, of:
    • E(b)
    • F(b)
    • A lidless

12 Hours Later

  • Ran gel of proteinase-K-ed final supernatant
    • Gel:
Lane Component Amount
1 1kb ladder 1uL (from stock of 1ug/uL)
2 p7308 9uL
3 c5.0.A (unbiotinylated barrel) wash-supernatant 38uL
4 c5.0.A (unbiotinylated barrel) final-supernatantc 38uL
5 5.0.8b wash-supernatant 38uL
6 c5.0.8b final-supernatant 38uL
7 c5.0."Eb" (inside biotinylation) wash-supernatant 38uL
8 c5.0."Eb" (inside biotinylation) final-supernatant 38uL
9 c5.0."Fb" (outside biotinylation) wash-supernatant 38uL
10 c5.0."Fb" (outside biotinylation) final-supernatant 38uL

    • Results:
      • NOTHING in final-supernatant lanes again, indicating either:
        • (A) Second trypsinization (not able to be visualized because ran the gel without enough buffer) worked, leaving nothing on the streptavidin for the proteinase K to have digested.
        • (B) Proteinase K did not digest the streptavidin to elute.
      • Wash lanes were also dim - did not Speedvac for long enough.
        • Also, gel was older and had been left out of fridge overnight.
          • Plan: Try again with proteinase K as eluent, as there were so many problems with this trial (ie. "Eb" and "Fb", trypsinization first two times around)

Folding design 6

  • Mixing pre-working stocks (c6.0.1, c6.0.2, c6.0.3, c6.0.4, c6.0.5)
    • To mix pre-working stocks from plates, pipet 10 μL of each of the appropriate oligos into 1.5 mL tubes.
    • List of pre-working and working stock oligos: Table_of_c6.0_Working_Stocks

(don't have time to complete. will do on sunday).