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)
• 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
• Next wednesday plan to perform zymo gel purification of part digest, ligation of part digest into plasmid digest, and cell transformation.

• Zymo Gel Purification, Ligation, Transformation (3/4)
• 1) Prepared four gel loading solutions (10 ul of EcoRI/BamHI digested PCR product with 3 ul of 2X loading buffer), ran prep gel, pictured gel (shown in gel pics), cut out 2 bands (both bands are for the Invasin-short part; differ in PCR annealing temp, lane 2 is for 50 C and lane 3 is for 45 C), and zymo gel purified part EcoRi/BamHI digests.
• 2) Prepared two ligation reactions of EcoHI/BamHI digests as follows: 6.5 uL ddH20, 1 uL T4 DNA Ligase buffer, 1 uL plasmid digest (pCA9495CA), 1 uL part digest, and 0.5 uL T4 DNA ligase, mixed + quick spinned.
• 3) Incubated ligation reaction on benchtop for 40 min, put on ice, and proceeded with transformation.
• 4) Prepared competent cell cocktail as follows: thaw a 200 uL aliquout of competent cells (in tubes with green line on lid), add 50 uL of H20, add 30 uL of KCM salts. Added 75 uL of the cell cocktail to the ligation, and mixed gently with pipette
• 5) Transformed cells by heat shock as follows: 10 min on ice, heat shock for 2 min at 42 C, back on ice for 1 min.
• 6) Plated on amp-selective agar plates (AC), and let incubate overnight at 37 C (incubator in room 327)
• Next, Tim or Gabriel plan to pick up colonies and grow in LB overnight.

• Miniprep purification of DNA (3/6)
• 1) Cannot proceed with this step; pick up colonies and grow in LB overnight step not done/overlooked. Will email Gabriel as a reminder for doing this step this sunday so as to do miniprep monday 3/9 before 10 am.
• 2) Asked by Gabriel not to continue with PCR to build Inv-long part (Construct 39) for which PCR failed. JAC will build this part.
• 3) Updated progress in google document.
• Next monday, plan to do miniprep