![]() Sterile water, 6 x 1 ml in microtubes for ligation and resuspending DNA.LB media, ~13 ml per group, put in 6 x 15 ml conical tubes.Sodium acetate 3M, 6 x 100 ul in microtubes.super competent XL1-blue cells, one 200 ul aliquot per group.only take out of -80☌ freezer when first group is ready, competency decreases with time on ice.aliquots of yeast tRNA from 109 Antibodies box.BamHI, 2 aliquots in PCR tubes in cold-boxes (~10uL each).T4 ligase buffer, has ATP so must be kept cold (~50uL in 3 eppendorfs).T4 DNA ligase, 3 aliquots in PCR tubes in cold-boxes (~10uL each).Refill burners and jars with 100% ethanol.Need 5/group = 60, 1L makes 30-40 plates, so need to make a couple liters. Freeze purified backbone and annealed inserts, make sure all phage plates are in incubator.Do not put out any reagents that are incomplete (e.g., do not put out PE that does not have ethanol added to it). Aliquot E4 and M13 phage stocks for dilutions, ~200 ul.20 ml sterile water in a few 50ml conical tubes.Need 6 LB plates/group taken out of -20C and 6 small sterile test tubes/group.Pipetmen, 5 ml pipettes and 50C water baths for phage plating.(SC: I microwaved 1', swirled, microwaved 30", and it was melted, then into 55C water bath) It's very important that the top agar be fully melted for the students. Microwave in water bath for 2', invert to distribute any unmelted parts, heat another minute. Put gels in boxes for running, remove combs, add ~500 ml 1X TAE.Need quiz (all quizzes 2 or 3 questions, 5 points).Check reagents and tubes in Qiagen Agarose Cleanup Kit.Check for phage stocks: M13K07 and another M13 phage called E4.Pour LB plates (2L), 6 plates per group, x 2 days = 72 plates.Need to pour 3 x 100 ml agarose gels for each day of lab, using one 10-well comb (thicker teeth) in each gel.Plate on LB+Kan, then set up one overnight of 3 ml LB + Kan/group. Need to set up ER2267 (NB271) so there will be cells for 1.2 ml/group.Remember to freeze away digests (-20C) before leaving lab for the night.Need to have plasmid for restriction digest, each group gest one miniprep of NB251.super competent XL1-blue cells (need one tube of 200 ul/pair of students).T4 DNA ligase and buffer for ligation mix.Qiagen kit for agarose clean-up (need 1/pair of students).restriction enzymes (BamHI,others on list of no cutters here ).Check volume/availability of needed kits, reagents:.Make 1L top agar, divide between autoclaved 250 ml bottles, 100 ml per bottle for reheating.Autoclave several racks of small and large test tubes.This is just to check that the phage stock is still active. The Kan is important to select for the F'. Streak out a plate on LB+Kan so you will have colonies to pick for liquid cultures before phage titration. This strain is in the lab collection as NB271. NEB titers M13K07 on their strain ER2267 cat#E4103S. ![]() Double-check volume of needed oligos: for cloning control, for sequencing.You do not have to pool since each group in the class will be given one tube for their cloning experiment that starts on Day1 of the module. Miniprep these for the vector DNA the next day. Make 16 overnight cultures (each 2.5 ml LB+Kan). Pour several liters of LB+Kan (~2L) and LB (~4L) plates. ![]()
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