Team:Hong Kong HKU/laboratorynotes

Lab note

We did kept a very detailed laboratory note during our experiments, for example, excerpted below is a single day experimental record of one of our teammate in the construction of pETFlexStack:

22/8
1. Restriction, Ligation, Transformation
• restricted pT7-pET28a and sig-pET28a MiniPrep products with NcoI;XhoI BglII;XbaI respectively in NEBuffer3-BSA for ~30min, subsequently treated with CIP from NEB for ~2h, 100ul reactions @ with 10ul DNA@. PCR-purified using QIAprep columns with water elution; gel analysis shows faint yet visible ds-breaked DNA of appropriate size compared with control; on a side note all MiniPrep products are heavily contaminated by RNA, as reflected on the huge NanoDrop results (>1000ng/ul)
• restricted incorrectly digested MB2016;MB2025-gel-purified products back in 20/8 with SalI in NEBuffer3-BSA, PCR-purified with water elution
• ligation was performed with appropriate compatible sites for all possible combinations, using ExpressLink T4 DNA Ligase from Invitrogen, 10ul reactions@, 30fmol vector, 90fmol insert, for ~10min at room temperature
• ligation products were diluted 5x with water and transformed 1ul diluent into ~50-60ul competent DH10B cells
2. Plasmid transformation
• transformed pMALc2 and pGEX4T1 into 20ul competent cells and plated on Ampicillin plates
3. Sequencing prep
• sent PT7-pET28a (colony 1&2) and Sig-pET28a (colony 1&2) for sequencing, took 15ul@ MiniPrep products for sequencing
4. Restriction analysis
• The inoculated colonies (1&2 for both) from PT7-pET28a and Sig-pET28a was MiniPrep-ed on 22/8, was subjected to BglII and XhoI digestion respectively in 20ul reaction, NEBuffer3-BSA, overnight digestion at 37°C
5. PCR
• Primers arrived.
• Did PCR MB3003;MB3008, using GoTaq Flexi G2 Polymerase from Promega, 3*100ul reactions with 2mM [Mg2+], 10ul@ for the primers and 2ul of template (BBa_K311004), annealing temperature 62°C, cool down overnight in machine
• PCR MB3002;MB3005, using LongAmp Polymerase from NEB, 3*100ul reactions, 10ul@ for the primers and 2ul of template (BBa_K311004), annealing temperature 58°C, cool down overnight in machine
Plan:
1. Gel analysis of restriction analysis of MiniPrep-ed plasmids
2. PCR-purify the PCR products, restrict and ligate into pET28a (with dephosphorylation), transform into DH10B
3. See plates front colonies in making PT7-Sig-pET28a, if have colonies, do PCR colony test; if not, redo ligation-transformation together with the BBa_K311004, or simply transform the saved overnight ligation mix at 16°C
4. Inoculate colonies to prepare a stock of pMALc2 and pGEX4T1 in the evening.

however for simplicity, we would provide a brief outline here.

June: lab meetings for the discussion of project ideas and approval
Early July: lab training and practical skills, primer ordering and experimental planning
Late July: cloning and construction of planned plasmids and systems
Early August: protein expression studies and extraction test; plasmid design and planning
Late August: protein purification with Ni-affinity column; plasmid construction
September: protein purification with gel filtration column, confocal microscopy; plasmid construction
October: protein purification fine-tuning