"

From 2013.igem.org

---

Revision as of 02:22, 5 October 2013

IMMUNOFLUORESCENCE

• Aspirate the medium from wells. Wash wells with PBS 1000 µl/well.
• Fix cells with prewarmed 4% Paraformaldehyde/PBS 500 µl /well for 15 min.at RT (add very slowly).
• Remove supernatant.
• Treat the cells for 15 min. at RT with prewarmed (37oC) TZN buffer 500 l/well (add very slowly). Slowly mix on shaker.
• Aspirate supernatant.
• Wash wells with PBS, 750 l/well, 5 min X 3 on shaker.
• Add Blocking Solution 500 l/well, incubate for 1 hr at RT. Mix slowly on shaker.

Blocking Solution (f) NGS 10% BSA (10%) 1% PBS-Tx 0.3%

Aspirate off the blocking solution. Add 100 l/ well 1st Ab. mixture. Seal the plate with parafilm, incubate at RT for 2 hr or o/n @ 4oC. Wash with PBS-TX 0.3%, 750 l/well, 5 min X 1 on rocking shaker. Wash with PBS 750 l/well, 5 min X 2 on rocking shaker. Add 100l/ well 2nd Ab. Work in dark from this point! Incubate the plate at RT for 1 hr. Wash with PBS 1ml/ well , 5 min X 3 on rocking shaker. Add 300 l/well TO-PRO-3 (1 M) (light sensitive!)

Incubate at RT for 15 min. Wash with PBS 1 ml/well, 5 min X 3 on rocking shaker. Add 1 drop of Prolong Gold Mounting Medium onto slides for each coverslip. Take out coverslip from the well. Invert and put on mounting soln. on the slide. Seal coverslip with nail polish. Let the coverslip dry. Slides can be stored at 4oC for a long time (at dark). Take images with confocal microscope.

Western Blotting Protocol Required Solutions 10x Running Buffer 30.3 g (0.25 M) Tris Base 144 g (1.92 M) Glycine 10 g (1%) SDS or appropriate for concentrated stock qs 1000 ml ddH2O Dilute 1:10 with ddH2O. pH will be 8.3 SDS-PAGE Gel and Western Blot Run the gel according to the instructions below. Refer to SDS-PAGE Gels on page 7 for details. 1. Measure protein concentration in duplicate using Bradford using a BSA standard curve. Run up to 70 μg/lane (expect 300-400 μg protein from a subconfluent 10 cm plate). 2. Mix extract 1:1 with Laemmli buffer and heat to 80-100ºC for 5 min (don’t boil too long, proteins get destroyed). 3. Run on an SDS-PAGE minigel until the blue front is at the bottom of the gel (refer to SDS-PAGE Gels for gel composition). Bio-Rad Mini-Gel Box Running Conditions: 75 V x 3 hrs (dye about in the bottom of the gel) Western Blot Protocol - de Lange lab 4 4. Transfer: Blot onto a nitrocellulose membrane. Pre-wet materials in transfer buffer. Stack in the following order: case (clear side) sponge Whatman paper membrane gel Whatman paper sponge case (black side) Place in the transfer apparatus with black side facing black). Bio-Rad Mini-Gel Box Electrotransfer: 90 V x 1 hr (use ice-pack to cool down the apparatus) 70 V x 2 hr (use ice-pack to cool down the apparatus) Transfer at 0.5 A-Hour (0.5 A for one hour, or 0.05 A for 10 hrs.) 5. Stain with 1x Ponceau S for a minute and destain in acidified H2O (2.3 l of ddH2O + 4 ml concentrated HCl). Wrap in plastic wrap and Xerox. Rinse in 1x PBS. 6. Block the membrane for 30 min in 20-30 ml 1x PBS + 5% non-fat dry milk + 0.1% Tween 20, in a small Tupperware dish on a shaker. 7. Incubate with primary antibody (see Antibodies for Shelterin Components on page 8) diluted in 2 ml 1x PBS + 5% milk + 0.1% Tween 20. Incubate o/n at 4ºC or 4 hrs in a seal-a-meal bag on a nutator at RT. Membranes can be stacked back to back in one bag. The primary antibody mix can be re-used (store at 4ºC.) 8. Wash 3 x for 5-10 min in ~50 ml 1x PBS + 0.1% Tween 20 at RT in a small Tupperware on a shaker. 9. Incubate with secondary antibody for 30 min to 1 hr at RT in 2 ml 1x PBS + 5% milk + 0.1% Tween 20 in a seal-a-meal bag on a nutator. Use Amersham antimouse or rabbit HRPO conjugated at a dilution of 1:2500. 10. Wash 3 x 10 min each in ~50 ml 1x PBS + 0.1% Tween 20 at RT in a small Tupperware on a shaker. 11. Rinse with ddH2O. 12. Detect protein with ECL kit (2 ml/membrane). In a separate tube, mix black and white ECL solutions in a 1:1 ratio. Then aliquot solution onto membranes and wait for 1 minute. Drain the ECL, wrap in plastic and expose to film.

PCR PROTOCOL  Protocol Reaction setup: 

We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (95°C).  Component 25 μl reaction 50 μl reaction Final Concentration 10X Standard Taq Reaction Buffer 2.5 μl 5 μl 1X 10 mM dNTPs 0.5 µl 1 μl 200 µM 10 µM Forward Primer 0.5 µl 1 μl 0.2 µM (0.05–1 µM) 10 µM Reverse Primer 0.5 µl 1 μl 0.2 µM (0.05–1 µM) Template DNA variable variable <1,000 ng Taq DNA Polymerase 0.125 µl 0.25 µl 1.25 units/50 µl PCR Nuclease-free water to 25 µl to 50 µl   Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.

Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin thermocycling. 

Thermocycling conditions for a routine PCR:  STEP  TEMP TIME  Initial Denaturation  95°C  30 seconds 30 Cycles 95°C 45-68°C 68°C 15-30 seconds 15-60 seconds 1 minute/kb Final Extension 68°C 5 minutes Hold 4-10°C  

General Guidelines:  1. Template: 

Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 μl reaction are as follows:  DNA Amount genomic 1 ng–1 μg plasmid or viral 1 pg–1 ng 2. Primers: 

Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 (http://frodo.wi.mit.edu/primer3) can be used to design or analyze primers. The final concentration of each primer in a reaction may be 0.05–1 μM, typically 0.1–0.5 μM. 3. Mg++ and additives: 

Mg++ concentration of 1.5–2.0 mM is optimal for most PCR products generated with Taq DNA Polymerase. The final Mg++ concentration in 1X Standard Taq Reaction Buffer is 1.5 mM. This supports satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.5 or 1.0 mM increments using MgCl2. 

Amplification of some difficult targets, like GC-rich sequences, may be improved with additives, such as DMSO (3) or formamide (4). 4. Deoxynucleotides:

The final concentration of dNTPs is typically 200 μM of each deoxynucleotide. 5. Taq DNA Polymerase Concentration: 

We generally recommend using Taq DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 μl reaction). However, the optimal concentration of Taq DNA Polymerase may range from 5–50 units/ml (0.25–2.5 units/50 μl reaction) in specialized applications. 6. Denaturation: 

An initial denaturation of 30 seconds at 95°C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, a longer initial denaturation of 2–4 minutes at 95°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute denaturation at 95°C is recommended. 

During thermocycling a 15–30 second denaturation at 95°C is recommended. 7. Annealing: 

The annealing step is typically 15–60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 45–68°C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm.  The NEB Tm Calculator is recommended to calculate an appropriate annealing temperature.

When primers with annealing temperatures above 65°C are used, a 2-step PCR protocol is possible (see #10).  8. Extension: 

The recommended extension temperature is 68°C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68°C is recommended. 9. Cycle number: 

Generally, 25–35 cycles yields sufficient product. Up to 45 cycles may be required to detect low-copy-number targets. 10. 2-step PCR: 

When primers with annealing temperatures above 65°C are used, a 2-step thermocycling protocol is possible.

Thermocycling conditions for a routine 2-step PCR:  STEP TEMP TIME  Initial Denaturation 95°C 30 seconds 30 Cycles 95°C 65-68°C 15-30 seconds 1 minute/kb Final Extension 65-68°C 5 minutes Hold 4-10°C    11. PCR product: 

The PCR products generated using Taq DNA Polymerase contain dA overhangs at the 3´–end; therefore the PCR products can be ligated to dT/dU-overhang vectors

Procedure for Spin Purification of His-tagged Protein

Note: The total volume of the 0.2mL, 1mL and 3mL column devices are 1.0mL, 8mL and 22mL, respectively. If a sample volume is greater than the column device, perform multiple applications and centrifugations until the entire sample has been processed. Be careful not to exceed the working capacity of the resin. The HisPur Cobalt Spin Columns also can be used for gravity-flow purifications. 1. Equilibrate column(s) to working temperature. Perform purifications at room temperature or at 4°C. 2. Remove the bottom tab from the HisPur Cobalt Spin Column. Place column into a centrifuge tube and loosen top cap.

Note: Use 1.5mL, 15mL or 50mL centrifuge tubes for the 0.2mL, 1mL and 3mL spin columns, respectively. 3. Centrifuge column at 700 × g for 2 minutes to remove storage buffer. 4. Equilibrate column by adding two resin-bed volumes of Equilibration/Wash Buffer. Invert column to mix. 5. Centrifuge column at 700 × g for 2 minutes to remove buffer. 6. Prepare sample by mixing 1 part protein extract with 1 part Equilibration/Wash Buffer. 7. Cap bottom of column. Add the prepared protein extract to the tube and cap top of column. Invert column to mix.

Note: For maximal binding, incubate for 30 minutes at 4˚C on an end-over-end rocking platform. 8. Centrifuge column at 700 × g for 2 minutes and collect the flow-through in a new centrifuge tube. 9. Wash resin with two resin-bed volumes of Equilibration/Wash Buffer. Centrifuge at 700 × g for 2 minutes and collect fraction in a centrifuge tube. Repeat this step two more times collecting each fraction in a separate centrifuge tube.

Note: If desired, perform additional washes. Monitor washes by measuring their absorbance at 280nm. 10. Elute His-tagged proteins from the resin by adding one resin-bed volume of Elution Buffer. Centrifuge at 700 × g for 2 minutes. Repeat this step two more times, collecting each fraction in a separate tube.

Note: If performing gravity-flow add two resin bed volumes of Elution Buffer to achieve proper flow characteristics. 11. Monitor protein elution by measuring the absorbance of the fractions at 280nm or by Coomassie Plus (Bradford) Protein Assay (Product No. 23236). The eluted protein can be directly analyzed by SDS-PAGE. To remove excess imidazole for downstream applications, use gel filtration (e.g., Thermo Scientific Zeba Spin Desalting Columns) or dialysis (e.g., Thermo Scientific Slide-A-Lyzer Dialysis Cassettes).

Protein Isolation With Sonication -Spin down from 50 mL of culture supernatant and resuspended in phosphate buffer saline containing PMSF in order to prevent proteis from proteases. -The mixture is then sonicated on ice eight times for 30 second with a 52% pulsed activity cycle ( Sonicator W 300). -Next, centrifuge the lysate for 30 min at 10,000 rpm to remove the cell debris. -Take supernatant including proteins

xCELLigence Cell Viability & Death Experiment 1. Count the cells and then seed cells in 16-well specific plate (Cell density changes according to cell line) 2. After 24 h, proteins/drug should be incubated with cells (For detailed cell thawing and seeding methods can be read.) The machine reads continuously the cell density increase/decrease.