SBB09Ntbk-CarlosRivera-Carpio

• Oligos (2/18)
• 1) Span tubes with DNA powder to pellet the powder
• 2) Added 270 uL ddH20 to 270 nmol of oligos to reach a final concentration of 100 uM
• 3) Aliquoted 1 uL and diluted ten-fold to 10 uM for PCR reaction recipe

• PCR (2/18)
• 1) Prepared in a PCR tube the following PCR reaction mix: 24 uL ddH20, 3.3 uL, 10X buffer "2", 3.3 uL dNTPs (2uM), 1 uL of forward oligos (10 uM), 1 uL of reverse oligos (10 uM), 0.5 uL of pAC-Tet-inv NoBglII
• 2) Added 0.5 ul Expand DNA polymerase "1"
• 3) Placed PCR reaction tube in PCR thermocycler programmed with C4K55 thermocycling protocol for an expected product of ~ 2.7 Kb

• Analytical Gel (2/23)
• 1) Prepared loading solution with 5 uL PCR product and 5 uL loading buffer
• 2) Load solution in gel lane 8
• 3) Ran gel until ladders were resolved
• 4) No visible band in lane 9 could be observed
• 5) Prepared a new loading solution with 8 uL PCR product and 5 uL loading buffer
• 6) Load solution in gel lane 11
• 7) Ran gel until ladders were resolved
• 8) No visible band in lane 11 could be observed

• PCR (2/23)
• 1) Prepared in a new PCR tube a reaction mix as in 2/18 making sure all added ingredient and all added volumes are correct.
• 2) Wrapped with tape PCR reaction tube, labelled tape with thermocycling protocol C4K55 label, handed in tape-wrapped-and-labelled PCR tube to TA

• Analytical Gel (2/25)
• 1) Prepared loading solution with 5 uL PCR product and 5 uL loading buffer
• 2) Load solution in gel lane 5
• 3) Ran gel until ladders were resolved
• 4) No visible/UV band in lane 5 was observed

• PCR (2/25)
• 1) Prepared 2 new PCR tubes each with a PCR reaction mix as in 2/18 but with 3.3 uL of DMSP instead of 3.3 uL of ddH20.
• 2) Prepared 2 new PCR tubes each with a PCR reaction mix in step 1 above but with 3.3 uL of dNTP (10 uM) instead of 3.3 uL of dNTP (2uM).
• 3) Wrapped with tape one PCR reaction tube from step 1 together with one PCR tube from step 2, labelled tape with thermocycling protocol C4K45 label (annealing temperature of 45 C).
• 4) Wrapped with tape the other PCR reaction tube from step 1 together with the other PCR tube from step 2, labelled tape with thermocycling protocol C4K50 label (annealing temperature of 50 C).
• 5) Handed in PCR tubes (4); Tim will ran PCR reactions.

• Analytical gel (2/27)
• 1) Prepared 4 loading solutions, ran analytical gel, and performed 4 regular Zymo cleanups
• 2) Prepared 4 loading solutions each with 5 uL PCR product and 2 uL loading buffer (6X)
• 3) Loaded solutions in gel as follows: a) lane 7 with solution for PCR product with 45 C annealing temperature and 2 uM of dNTPs; b) lane 8 with solution for PCR product with 50 C annealing temperature and 2 uM of dNTPs ; c) lane 9 with solution for PCR product with 45 C annealing temperature and 10 uM of dNTPs; d) lane 10 with solution for PCR product with 50 C annealing temperature and 10 uM of dNTPs
• 4) Ran gel until ladders were resolved
• 5) Lanes 7 & 8 showed visible/UV smear bands; DNA ladder not very well resolved; main band in lanes 7 & 8 appear to correspond to the expected 2.7 Kb. No visible bands in lanes 9 and 10 were observed (gel picture taken and loaded)
• 6) Next monday will use the Zymo cleanup products for the 2 PCR reactions with the expected product. Will also ran analytical gel to confirm above results.

• EcoRI/BamHI Digest of PCR products" (3/2)
• 1) Prepared in a fresh tube a digest reaction for each of the 2 PCR reactions with the expected product (corresponding to annealing temperatures 45 C and 50 C) as follows: 8 uL of PCR product, 1 uL of NEB buffer 2, 0.5 uL EcoRi, 0.5 uL BamHI
• 2) Incubated at 37 C on the thermocycler for 1hr
• 3) Prepared in a PCR tube another digest reaction for each of the 2 PCR reactions (as in step 1 above) and incubated at 37 C on thermocycler for 1hr
• 4) Prepared one gel loading solution (3 ul of PCR product with 45 C for annealing temperature with 1 ul of loading buffer) and ran analytical gel. Verified 2.7 Kb band in lane 5 by comparison with 1 Kb DNA ladder.
• 5) Performed regular Zymo cleanup to digested reactions (4) as follows: tranfer PCR product digest into the Zymo column inside a collection tube; spin through, discard waste; add 300 uL of Zymo ADB buffer (brown bottle); spin through, discard waste, add 200 uL of PE buffer; spin through, discard waste; add 200 ul of PE buffer; spin through, discard waste; spin for 90 seconds, full speed to dry; elute with 8.5 uL of water into a fresh Eppendorf tube
• 6) Next wednesday plan to perform zymo gel purification of insert digest, ligation of insert digest into vector digest, E-coli transformation, and picking of colonies.