Lab Protocols

Yeast transformation Cell Competency and Transformation Protocol (Schiestl & Gietz) 1. Pipette 5 ml of dropout media into a sterile 17 x 100 mm, 14 ml polypropylene, round bottom tube (e.g. Falcon). Inoculate media with yeast from a growing colony. Shake in 30°C incubator at 250 rpm overnight. You may want to inoculate several tubes -- use the one which grows fastest for the remainder of the procedure. 2. Vortex the yeast tube (to disaggregate HF7c yeast strains) and add the contents to a sterile 125 ml flask containing 45 ml of YPD. Shake the flask containing the 50 ml mixture in a 30°C incubator at 250 rpm for 5-12 hrs (optimal incubation time is empirically-derived). 3. Pour the contents of the flask into a sterile 50 ml Corning centrifuge tube and place the tube into a fixed-angle rotor within centrifuge. Balance tube and spin at 7000 rpm for 5 min. Pour off supernatant. 4. Resuspend pellet in 50 ml of ddH2O by vortexing. Repeat centrifugation at 7000 rpm for ...

Competent cells Preparation 1. Pick one colony off fresh DH5() plate into 2.5 ml LB supplemented with 25 l 1M MgSO4 (10mM final conc.) 2. Shake at 37°C overnight and until use 3. Do a 1:500 dilution by inoculating 100 ml of SOB in 1 L flask with 200 l of o/n DH5(), record start time 4. Spec starting at 3 hours after start time to an OD550 0.15 to 0.3 or "eye spec" every 20 minutes starting at 3 hours after start time. 5. Collect in two pre-cooled 50 ml orange cap c/f tubes and incubate on ice 15 min 6. Pellet in the hermle swinging bucket rotor at 2500 rpm for 5 min at 4°C with no brake, drain pellet thoroughly 7. Resuspend in RF1 in 1/3 original volume(30 ml) by gently pipetting with DNA tips 8. Pellet in the hermle swinging bucket rotor at 2500 rpm for 5 min at 4°C with no brake, drain pellet thoroughly 9. Resuspend by gently swirling in RF2 in 1/12.5 original volume (8 ml) and incubate on ice 15 min 10. Aliquot into pre-cooled tubes...

Sequencing Procedure Plate Preparation 1. If new plates, mark the treated side of the plate with an RVS using the diamond pencil. 2. Using Alconox soap and glass sponge, clean larger (non-treated) plate with small circular motions including corners in dish sink using styrofoam box for support. 3. EtOH and wipe clean with kimwipe. 4. Lay on two green test tube racks and EtOH/kimwipe dust off. 5. Place side spacers with one foam end. 6. Repeat cleaning as in step 1 & 2 on treated glass (smaller plate). 7. Treated plate can be used 3-5X between treatings with Rain-X(see below). 8. Check treated plate for dust and clean. 9. Place plate treated side down onto of spacers. 10. Position bottom spacers so seal if formed. 11. Place one clip on each side and two clips on each corner, 5 clips on each side total. Rain-X Plate Treatment 1. Clean smaller treated plate as usually 2. Squirt a small amount of Rain-X onto kimwipes. 3. Apply t...

Seqencing reactions (using Amersham ThermoSequenase Kit) 1. a) Get full tub and bucket of ice, b) get isotope out if in -20°C 2. Get dGTP or dITP master mix buffer and reaction buffer out and thaw on ice 3. Thaw primers on ice, they need to be 2 pmol/l 4. Label 4 PCR tubes per sample in the following order and color code: Base Letter Marker Color Description A black all colors C red crimson G green T blue teal 5. Label 4 colored tubes for termination mixes and _X_ colored tubes for sample reaction mixes 6. Get dNTPs out to thaw 7. Do termination mix calc. using kit quick card as preparing for (n + 1) samples 8. Do reaction mix calc. (normally 1 l of mini prep is used, so adjust H2O) 9. Transfer 2.5 l termination mix to each appropriate tube (left to right) 10. Transfer 4.5 l reaction mix to each appropriate tube (front to back) and mix sol'n with pipet tip Tub diagram: T T T T T ...

Library screen Day 1 * Start overnight of LE392 in LB with 10mM MgSO4 Day 2 * Make 12 plates for lifts using 10,000 plaques per plate dilution * Combine 10 l of dilution to be plated with 100 l of LE392 in 1.7 ml bullet * Warm (no shaking) at 37°C for 15 min * Add phage/LE392 to 3 ml lambda top agarose cooled to 50°C * Working quickly, vortex for 10 seconds and pour all 3 ml onto a pre-warmed (37°C) NZY or LBM plate; rock plate to spread agarose out over entire surface * Once plates are solidified, incubate plates at 37°C for 6 hours * Prepare probe following probe labeling protocol * After 6 hours, place plates in single layer for 1 hour at 4°C to chill * While chilling, label nylon circles with pencil or sharpie * Prepare denat. and neutral. filter paper/saran wrap stations * Have small amount of 2xSSPE in square glass dish for rinsing * Have filter paper for drying circles and filter paper size of crosslinker to p...

Library titer for ZAP cDNA Want the dilution that gives 10,000 plaques per 100 mm diameter plate Keep all dilutions made from the library for later use Day 1 Streak LE392 onto a NZY or LBM plate and grow o/n at 37°C Day 2 Start an o/n from the fresh plate of LE392 in LB with 10mM MgSO 4 Make the following dilution series: Combine 10 l of dilution to be plated with 100 l of LE392 in 1.7 ml bullet Warm (no shaking) at 37°C for 15 min Add phage/LE392 to 3 ml lambda top agarose cooled to 50°C Working quickly, vortex for 10 seconds and pour all 3 ml onto a pre-warmed (37°C) NZY or LBM plate; rock plate to spread agarose out over entire surface Once plates are solidified, place at 37°C o/n Day 3 Count plate(s) that show 30-300 plaques per plate Back calculate to determine library concentration Determine dilution to use for 10,000 plaques per plate

Probe counter (Beckman -- across from Denell's lab) Remove cover from printer From center tower the machine goes clockwise From left to right put in stop, sample(s), and user tag on #8 (inserts into holder only one way) Close door and push auto (green) Will print report automatically When finished put printer cover back on so it does NOT cover fan vents

Probe labeling using decamer oligos Standard Reaction Final Volume of rxn is 50 l. Make sure water added to DNA in step 1 is adjusted for the amount of isotope used in step 2. Components in -20°C box: "oligo labelling". Step 1: 1 l DNA (approx. 100ng/ l) 29 l ddH 2 O - heat 5 min at 95°C - cool on ice - spin Step 2: add 10 l 5X decamer buffer 2 l BSA (10mg/ml) 2 l dNTPs minus dCTP (0.5 mM) 0.5 l exo(minus) Klenow 5 l P 32 dCTP (fresh) --------------------------------------- 50 l total volume Transfer tube to 37°C heat block for 30-45 minutes Add 50 l TE(10/1) to reaction and transfer to a dried (2 min at medium in clinical) G-50 column Spin 2 min on medium Add 100 l TE to column to rinse Spin 2 min on medium Transfer sol'n to new labelled tube and estimate volume Transfer 1 l to small piece of filter paper for counting and allow to dry Count probe on Johnson's machine with user #6 card(see below)...

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

cDNA phage stock and in vivo excision Phage stock Start an overnight of XL1-Blue from a fresh plate in LB (no antibiotics) Add 100 l XL1-Blue o/n to 3 ml of lambda top agarose at 50°C Vortex for 10 sec Quickly pour onto pre-warmed LB plates, tilt plate to spread evenly Spot 1-3 l (usually use 2 l) of phage stock onto plate using pipettor; can do 4-6 cDNAs on one plate if careful Incubate o/n at 37°C to get large plaque Transfer plaque using big end of pasteur pipet to 0.5 ml SM in micro c/f tube Add one drop of chloroform Cap well and vortex Store at 4°C, do not freeze

Qiagen cosmid prep Grow 25 ml cells in appropriate antibiotic overnight in a flask. Spin down cells at 6000 rpm in Hermle at 4°C for 10 min. Discard supernatant and resuspend in 0.9 ml P1 Divide resuspended cells into three 1.5 ml microfuge tubes Add 0.3 ml P2 to each tube (per Qiagen protocol). Add 0.3 ml P3 to each tube (per Qiagen protocol). Incubate on ice for 15 min (per Qiagen protocol). Pellet precipitate, spin 15 min at 4°C (per Qiagen protocol). Prepare Qiagen mini column (per Qiagen protocol). Pool supernatant from the three tubes onto ONE Qiagen column. Proceed with Qiagen mini protocol Resuspend DNA in 25-40 l TE, depending on size of pellet.

1. Grow 3-4 tubes of each cosmid in 5 ml media overnight 2. Harvest a total of 4 ml of each tube separately in 2 ml centrifuge tubes 3. Resuspend completely in 200 l Solution I* 4. Add 400 l Solution II* and invert until solution clears 5. Add 300 l Solution III* and invert gently 6. Hold on ice for 10 min 7. Spin 10 min at 4°C and transfer s/n to new tube 8. Phenol/chloro/IAA one time with equal volume 9. Spin 1 min at 4°C and transfer upper layer to new tube 10. Ppt with 0.8 vol isopropanol 11. Spin 20 min at 4°C 12. Remove s/n and allow pellet to dry 13. Resuspend in ~ 50 l of TE + RNase 14. Cosmids => Digest 5-10 l with either EcoRI or Hind III *refer to Alkaline Lysis Solution I, II, or III recipes in card box

Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust ...