Lab Protocols

Materials: -primers (24 and 25 are for slitrk 3) -cDNA (40 hour/90 hour) -10mM dNTP (nucleotides) -buffer 5x -water -enzyme (cooler in freezer) Combine in a PCR tube (50 microliters of solution): 1. 35.5 uL of water 2. 10uL 5x buffer 2. 1uL of JR 24 3. 1uL of JR 25 4. 1uL of dNTP 5.1uL of cDNA 6. 0.5 uL of Fusion hot start enzyme Add water to the PCR tube first. Make sure you use the PCR tubes because they fit in the machine. After all contents have been added, flick PCR tube and centrifuge for 4 seconds. To Run the PCR Machine: The power switch is on the back. The lid will click, and then the wheel should be twisted until you feel resistance. Program: JRRTPCR enable heated lid The reaction takes several hours. The machine with have a block temp of 4 degrees when done. Remember to turn off the machine when finished using it.

Day 1: see "Bacterial Transformation by Heat Shock" **Day 2: Starter Cultures and Inoculate into Large Bacteria Flasks** NOTE: Starter culture is preferable, but not required; can immediately go to larger flasks if you want to STARTER CULTURE: 1. Aliquot 2ml of the LB+Amp solution (0.02g Amp/200ml LB) into 14ml round bottom tubes 2. Using a yellow pipet tip, pick up an isolated large colony and drop the tip into the correct tube 3. Cap and place into incubator (37C) with shaker set to 200rpm for ~6 hours Note: At this point, can refrigerate starter cultures until ready to inoculate and do midi-prep kit INOCULATION: 1) Add Amp to LB broth at 100ug/ml (so, for 500ml of LB broth, add .05g Amp) 2) Add 100ml of LB+Amp into a 1L flask for every plasmid you are inoculating 3) Transfer 200ul of bacteria from starter culture into corresponding flask 4) Cover each 1L flask with aluminum foil 5) Incubate for 12-16 hrs in Dana Incubator (if necessary, use paper towels t...

Additional instructions for Expand (Roche/Boehringer-Mannheim) Long Template PCR Master Mix I Master Mix II template enzyme primers buffer dNTPs ddH 2 O to 25 l ddH 2 O to 25 l Notes: Preheat PCR machine to 94°C using instant key Mix the above two for 1 reaction F.V. 50 l Use thin wall 0.2 ml PCR tubes Vortex to mix thoroughly Spin pulse Overlay with 30 l oil even with hot bonnet Master mix I can be mixed in thin wall tube when few samples Master mixes of I or II can be done for large # of reactions

Standard PCR Conditions for Herman Lab (not for Expand PCR) Component Suggested range We use for 50 l rxn: __ ul of __ stock primers 20-500 pM 50 pM 2.5 of 20pM/ l buffer 1X 1X 5 of 10X dNTPs 20-500 M 200 M 5 of 2 mM MgCl 2 1-9 M 2.5 M* 5 of 25 M Taq 1-2.5 units 2 units 0.5 l DNA template 10 5 -10 6 (units?) ~50 ng genomic ~10 ng plasmid N/A * unless it's in the buffer or you are varying the [Mg 2+ ] Master Mix I Master Mix II annealing 5°C below lowest T m of primer or close to that, no lower than 40°C extension Temp: 72°C, Time: 1 min/Kb of expected product, 10 min on last cycle denaturation Temp: 95°C, Time: 5 min on initial cycle and 30 sec to 1 min on rest number of cycles 25-30, usually 30 Notes: Primer stocks are 200 pmoles/ l in TE pH 8.0 Use sterile ddH 2 O for dilutions and reactions Filter tips should be used when getting into stocks (dNTPs, primers) Make 100 l dilutions of primers in 0.6 ml tube