Team:USyd-Australia/Project

From 2014.igem.org

(Difference between revisions)

Revision as of 06:48, 19 September 2014

Project Protocols

All protocols and lab procedures used during our iGEM project is shown below.

Agarose Gel Electrophoresis
Gibson Assembly	Heat-shock Transformation
Ligation	PCR
Plasmid Mini Prep	Common Solutions
Competent Bacterial Cells	QiaQuick DNA Purification
Restriction Digest	SDS-PAGE

Agarose Gel Electrophoresis (Safety)

Add 1 g of agarose to 100 mL TBE.
Gently mix and microwave heat until dissolved.
Prepare gel tray. Depending on the tank, use masking tape or end-formers to seal the two ends of the gel tray. If using end formers, seal the ends by pipetting a small volume of gel solution into the contact between the end formers and tank and allow to set.
Allow gel solution to cool before adding 0.5 µL of Gel Red and pouring into gel tray. Alternatively, exclude the Gel Red and stain with Ethidium Bromide after running the gel.
Insert the well comb into the appropriate position in the gel.
Once set remove the comb, tape/end formers and place into electrophoresis chamber and submerge in TBE.
Mix samples with loading buffer (use 1:5 ratio of loading buffer to sample) and pipette into wells.
Apply 120-180V, depending on time constraints and desired resolution.

Back to top

Enzymatic Ligation Assisted by Nucleases (ELAN)

ELAN

150U XbaI
150U SpeI-HF
2000U T4 DNA Ligase
1X NEB Buffer 4
1mM ATP
2ug Clean PCR product

Back to top

Gibson Assembly

NEB website

Add 10 µL 2x Gibson Assembly Reaction Buffer to each tube.
Add the DNA fragments to be assembled; for a 2-3 fragment assembly use 0.02-0.5 pmol, and for a 4-6 fragment assembly use 0.2-1 pmol.*
Add 10 µL commercially provided positive control DNA to the positive control tube.
Make all tubes up to 20 µL*** with ROW.
Incubate samples in a thermocycler at 50°C for 60 minutes. Following incubation, store samples on ice or at -20°C for subsequent transformation.

	aldA	p450	tetR
Water	1.75 µL	1 µL	2 µL
~ 2000bp pSB1C3 (at 25ng/µL), 0.15pmol/µL	3 µL	3 µL	4 µL
~ 600bp gBlocks (at 20ng/µL), 0.5pmol/µL	2 x 2 µL	1 x 2 µL	-
~ 600bp gBlocks (at 40ng/µL), 1.0pmol/µL	-	4 x 1 µL	-
~ 400bp gBlocks (at 20ng/µL), 0.75pmol/µL	1.3 µL	-	2 x 2 µL
NEB Gibson Assembly Master Mix	10 µL	10 µL	10 µL
	20 µL	20 µL	20 µL

Back to top

Heat-shock Transformation (Safety)

Set-up a heat-block or water-batch at 42°C.
Retrieve aliquots of cells from -80°C freezer. Thaw on ice.
Add 1 pg-100 ng (1 µL - 5 µL) of plasmid DNA into cell suspension.
Incubate tubes 42°C for 30-45 seconds.
Return to ice and quickly add 1 mL of LB-broth. Incubate tubes for 1 hour on the 37°C shakers.
Spread-plate 100 µL of cells on LB containing the appropriate antibiotic.
Alternatively, try diluting or concentrating cells before plating. For instance, centrifuge the tubes, pour off supernatant, resuspend cells in the final drop remaining in the tube and spread-plate the last 100 µL of cells.
Incubate overnight at 37°C.

Back to top

Ligation

Digest plasmid e.g. Use 250 ng in digest volume of 100 µL although there is a wide acceptable range for this. (See restriction digest protocol)
At the same time as step 1, digest PCR product e.g. Use 1 µg in 100 µL digest. (See restriction digest protocol)
Combine on ice 2 µL of T4 ligase buffer (10x), 8 µL purified insert, 8 µL of purified vector, and 2 µL of T4 DNA ligase. Make sure ligase enzyme remains in ice at all times.
Split solution between two reaction tubes. Incubate one tube for an hour at room temperature. Incubate the other overnight at 4°C.
Throw out ligase buffer. Do not return to -80°C freezer.

Back to top

PCR

PCR of multiple samples is sped up significantly by making a master mix; the volumes below are for one 50 µL reaction, but the volumes would be multiplied by the number of reactions required and mixed together in a single tube.
- 5 µL 10x Pfu buffer/NEB Thermopol Buffer
- 1 µL dNTPs at 10 uM (final 200mM)
- 1 µL primer (F) (final 0.5-1uM)
- 1 µL primer (R) (final 0.5-1uM)
- 40.5 µL sterile MQ water
- 0.5 µL Pfu/Taq polymerase
Aliquot master mix into PCR tubes (49 µL) then add 1 µL of the template DNA. Alternatively, if performing a colony PCR, aliquot 50 µL of master mix into a tube and resuspend cells directly from the plate into the tube. (Only dip the toothpick 3-5 times in the master mix).
Start the thermocycler at the setting desired. Cycles must have a peak high enough to denature template DNA, a trough low enough to allow annealing of primer pair, followed by an intermediate stage for the optimal polymerase activity.

Back to top

Plasmid Mini Prep - 100 mL (Safety)

Pellet 100 mL culture in 2 x 50 mL Falcon tubes. Resuspend cells in 4 mL TE buffer, combine resuspended pellets in one tube.
Add 8 mL lysis solution (SDS-OH), mix well by inversion and shaking(~10 sec). Should go viscous. Leave at room temp for 15 min.
Add 6 mL ice-cold precipitation solution (K.Ac). Shake briefly – essential that K.Ac is thoroughly mixed in. Viscosity should disappear, and white precipitate appears. Keep on ice 15 min.
Spin at top speed (4000 rpm) in cold Centaur centrifuge for 15 min. Recover tube immediately and handle gently (pellet is soft and easily resuspended). Pour supernatant into new tube. Try to avoid the white junk, but don’t worry if little bits of it get transferred.
Add an equal volume isopropanol (~15 mL), mix well ice 15 min.
Spin at top speed (4000 rpm) in Centaur centrifuge (doesn’t need to be cold) for 15 min, pour off supernatant, keep pellet.
Add 10 mL 70% ethanol to pellet, resuspend by brief vortexing, leave for 5 min at room temp. Spin 15 min in Centaur centrifuge (doesn’t need to be cold). Pour off supernatant again.
Drain off excess supernatant by gentle tapping on paper towel, then heat at 50°C for approx 10 min to remove ethanol and isopropanol.
Redissolve pellet in 2 mL TE with mixing (tapping tube ~ 1 min , don’t vortex too much from this point onward). Can heat if necessary (eg. 50°C, 10-30 min). Note that plasmid DNA is much more soluble than chromosomal DNA, and dissolves preferentially. Solution should look slightly viscous (traces of chromosomal DNA still present).
Split prep into 2 x 1 mL in Eppi tubes. Extract each tube with phenol: chloroform: isoamyl (PCI), as follows. Suck up 500 µL PCI from under the aqueous layer in the reagent bottle, transfer to Eppi tube. Vortex for ~5-10 sec until a uniform milky white emulsion is obtained. Centrifuge 5 min. Transfer top phase (aqueous) to a new Eppi tube. Discard bottom phase (PCI) into phenol waste. Avoid the white junk at the interface between phases.
Repeat solvent extractions using 500 µl chloroform:isoamyl (CI, µl) per tube, as described for PCI above. After mixing & centrifugation, keep top aqueous phase, discard bottom CI phase into waste.
Split DNA prep into 4 equally sized aliquots (~400 µl each) Precipitate DNA by adding 1/10-volume 3M Na-acetate (~40 µl) to each tube, and then add 2 volumes cold ethanol (~1 ml). Incubate >2 hr at -20°C (overnight is fine).
Spin for 10 min, drain off supernatant, rinse pellet with 70% ethanol (as above, but using 500 µl 70% EtOH), drain excess EtOH off, then dry 10 min at 50°C.
Redissolve plasmid in 100 µL EB. Expected yields range from approx 10 µg plasmid per ml culture (eg pUC/pGEM) down to 0.2 µg plasmid per mL culture (eg. RSF1010). Final expected DNA conc. may range from approx 10-500 ng/µL.

Solutions: (see Sambrook Appendix 1)

Back to top

Preparation of Common Solutions

LB media

Mix 10 g Tryptone, 5 g Yeast extract, 5 g NaCl and 1 L of water, or use the same ratio in the required volume.
Autoclave the solution to sterilise.
Antibiotics can be added to make the media selective for antibiotic resistant cells. This must be added after autoclaving.

Potassium-phosphate Buffer

^-

Add 2.27 g/L K₂HPO₄ to 0.95 g/l KH₂PO₄.
Make up to 1 L with water.

Back to top

Preparation of RbCl Competent Bacterial Cells (Safety)

Streak E.coli strain onto LB agar and incubate overnight at 37°C.
Inoculate 5 mL LB broth with a single colony from LB plate. Incubate at 37°C with shaking.
Measure OD₆₀₀ of overnight culture. Aseptically inoculate 100 mL LB in 500 mL Schott Bottle with enough culture to obtain OD₆₀₀ of ~ 0.05.
Grow cells at 37°C, shaking until culture reaches OD₆₀₀ of ~ 0.5.
Aseptically transfer to centrifuge bottles and keep on ice.
Harvest cells at 4000 rpm, 4°C for 10 min in cold centrifuge.
Working in cold room with cells in ice, pour off supernatant and remove residual liquid by pipetting.
Resuspend pellet gently in 33 mL RF1 solution. Mix by pipetting up and down.
Incubate on wet ice for 1 hour.
Pellet bacteria at 4000 rpm 4°C for 10 min.
Again remove supernatant (work in cold room).
Resuspend pellet in 8 mL RF2 solution and mix.
Incubate on wet ice for 15 min.
Working quickly, aliquot 110 µL of cell suspension into pre-chilled microcentrifuge tubes. Once dispensed, collect all tubes and store in -80°C freezer.

Back to top

QiaQuick DNA Purification Kit

Mix your DNA sample with the appropriate buffer, in the appropriate ratio: - DNA < 4 kb: Mix 1 vol sample with 3 vol of QG buffer. - DNA > 4 kb: Mix 1 vol sample with 3 vol of QG buffer + 1 vol isopropanol. - PCR products: Mix 1 vol sample with 5 vol PB buffer.
Load the mixture onto a Qiaquick spin column (purple) and spin 30 sec. Discard the flow-through, and replace spin column in the catch tube. The spin column will hold a max. of 800 µL sample and has a max. binding capacity of approx 10 µg DNA. You can wash through multiple 800 µL aliquots of DNA+QG if you have a lot of sample, so long as the total amount of DNA added doesn’t exceed approx 10 µg per column.
Add 750 µl of buffer PE to the column, allow to sit for ~2 min, then spin 30 sec, discard flow-through, replace spin column in catch tube.
Spin again for 30 sec to remove all traces of PE from the column. Discard both the flow-through and catch tube, and transfer spin column onto a clean Kimwipe. Leave the column lid open. Transfer Kimwipe to 50°C incubator box, and allow to dry for 10 min.
Transfer spin column to a sterile 1.5 mL Eppi tube, and add 50 µL* of EB buffer (5 mM Tris, pH 8) to the centre of the spin column – ie on the membrane, not the walls of tube. Allow to sit for 5 min. Spin 30 sec, retain Eppi tube with DNA solution in EB, discard spin column.

Back to top

Restriction Digest

Add 5 µL of appropriate 10x buffer to a microcentrifuge tube. The buffer choice depends on the restriction enzyme used and can be checked from readily available tables.
Add DNA sample solution (~1 µg DNA).
Make tube up to 49 µL with reverse osmosis water.
Add restriction enzyme; 10 U activity is sufficient, which is normally the activity of 1 µL restriction enzyme solution. The restriction enzyme volume should not be more than 1/10 of the total reaction volume.
Incubate for an hour at the optimal temperature for restriction enzyme activity; again, this should be checked from relevant data tables.

Back to top

SDS-PAGE (Safety)

Sample Preparation

Inoculate 10 mL of LB broth with each culture. Leave on shaker at 37°C overnight.
Spin liquid cultures at top speed (4000 rpm) in cold Centaur centrifuge for 5 min at 4°C.
Remove supernatant and resuspend cells in 1 mL elution buffer.
Lyse cells using glass beads in a beadbeater (5.5 m/s, 30 s).
Spin for 5 min to pellet cell debris.
Transfer supernatant to clean microfuge tubes and use in SDS-PAGE or store at -20°C.
For use in SDS-PAGE, add 2x loading buffer (0.06 M Tris, 2% (w/v) SDS, 5% (v/v) ß-mercaptoethanol, 10% (v/v) glycerol 0.1% (w/v) bromophenol blue, pH 6.8) to equal volume of sample.
Heat each sample to 99°C for 5 min.

Gel preparation:

Prepare the resolving gel first by adding reagents as below.
- ROW – 3.4 mL
- Buffer – 2.5 mL
- 30% acrylamide/bisacrylamide – 4 mL
- 10% SDS – 0.1 mL
- 10% ammonium persulfate solution – 50 µL
- TEMED – 5 µL
After adding the TEMED, immediately pipette the liquid mixture into the gel apparatus until it reaches approximately three-quarters of the way to the top.
Pipette ~2 mm ROW onto the top of the gel and leave to set.
Once the resolving gel has set, prepare the stacking gel as below.
- ROW – 3.05 mL
- Buffer – 1.25 mL
- 30% acrylamide/bisacrylamide – 0.65 mL
- 10% SDS – 0.05 mL
- 10% ammonium persulfate solution – 25 µL
- TEMED – 10 µL
After adding the TEMED, pour off the water on top of the resolving gel and pipette in the stacking gel mixture until it reaches ~5 mm from the top. Add-well formers and allow to set.
Once the gel has set, add running buffer (0.025 M Tris, 0.192 M glycine, 0.1% w/v SDS). There are two buffer levels; in the inner chamber buffer should be added so that it just covers the wells, and in the outer chamber it should be added to ~1-2 cm above the bottom of the gel. Remove the well-former.
Load protein standard ladder and different volumes of treated cell lysates to each well as appropriate.
Run gel for ~40 min at 200 V. Monitor progression of the experiment by migration of the tracking dye.
Once the gel has finished running, switch off power and remove the gel from the apparatus.
Stain with Coomassie blue (0.005% w/v in acetic acid (7% v/v) and methanol (10% v/v)) rocking gently for 15 min.
Pour off the staining solution and add destaining solution (acetic acid (10% v/v) and methanol (10% v/v)). Rock gently with the destaining solution for 30 min.
Pour off destaining solution and add water. Image the gel.

Back to top

@@ Line 41: / Line 41: @@
 <br><br><br>
 </html>
-{{Team:USyd-Australia/ProtocolHTML}}
+{{:Team:USyd-Australia/ProtocolHTML}}