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Team:VIT Vellore/Documentation
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Revision as of 20:49, 5 October 2011 by A.Sivakumar (Talk | contribs)
Reaction Mixture-
- 1µl of samµple DNA template
- 2µl of 10X buffer
- 1.2µl of MgCl2
- 2µl of dNTPs
- 0.5µl of KOD DNA polymerase
- 2.0µl of forward and reverse primers each
- 9.3µl of water to bring reaction volume to 20ul
Thermocycler-
- 94*C for 5 mins, initialisation
- 30 cycles of:
• 94˚C for 1 min, Denaturation
• 55˚C for 1 min, Annealing
• 72˚C for 3min, Elongation
- 72˚C for 10 mins, Final Elongation
- 4˚C indefinitely, Final Hold
Agarose Gel Electrophoresis
- The agarose gel is prepared with BromoPhenol Blue added as tracking dye.
- The lengths of each part are noted down.
- 20ul of the samples and 7ul of an appropriate ladder are loaded and kept for electrophoresis at 100V for 45 mins.
- After running, the gel is taken to the gel doc machine, and the image under UV transillumination is captured.
Restriction Digest
Component Volume Water, nuclease-free 15 µl 10X FastDigest® buffer 2 µl DNA 2 µl (up to 1 µg) FastDigest® enzyme 1 µl Total volume 20 µl
Gel elution
Materials:
1. Agarose Gel
2. Tris-EDTA (T.E)
• 10mM Tris-HCl pH 7.6
• 1mM EDTA
3. Isopropanol/ phenol
4. Chloroform
5. Ethanol
6. Sodium acetate
Procedure:
1. Run DNA on "Low melt" agarose gel
2. After band have separated, visualize band on UV box, cut band out
3. Add 100ml of T.E to band, crush, heat to 65oC for approx. 5 min, add 200ml of phenol/ isopropanol, vortex, heat 65oC 3 min., vortex
4. Microfuge 5 mins, remove supernant
5. Add 100ml of T.E. to phenol/isopropanol, vortex, heat 65oC 3 min, vortex
6. Microfuge, pool supernants.
7. Chloroform extract (approx. 400 ul), microfuge 3 min.
8. EtOH precipitate, adding 1/10 vol. 3M Na0Ac, 2.5 vol. EtOH.
9. -20oC 1-2+hrs; spin 30 min., dry down, bring up in suitable vol 0.1X T.E.
Precautions:
Ligation of Vector and Insert
Let the mixture stand at Room Temperature (25oC) for 5 min.
The ligation is complete.
Perform an AGE to confirm the success of the reaction.
Transformation and competent cell preparation:
Chemical competent cell preparation
Materials:
1. Culture media
2. LB Broth
3. CaCl2
4. glycerol
Procedure:
1. Grow bacteria.
2. Inoculate 1:100 into LB.
3. Grow until OD @ 600 reaches 0.4-0.6.
4. Take a 2ml aliquot.
5. Centrifuge at 6000rpm for 8 min at 4˚C.
6. Resuspend the pellet in 800µl ice cold CaCl2.
7. Chill on ice for 30 min.
8. Centrifuge at 6000rpm for 8 min at 4˚C.
9. Re-suspend the pellet in70µl ice cold CaCl2 + 10µl glycerol.
9. Freeze the cells.
Transformation
o Competent cells had been prepared previously and stored.
o The competent E.coli cells are taken and thawed on ice.
o Next the DNA (5µl) is added to the cells and undergo heat shock.
o The mixture is incubated at 42oC for exactly 90 seconds and then immediately placed on ice.
o Left for 1 hour incubation.
o The transformation is complete.
o The transformed cells are plated and screened using a suitable antibiotic.
Preparation of Antibiotics
o Take ampicillin stock and dilute to a concentration of 100mg/ml with sterile water.
o Mix thoroughly and using a sterile syringe, withdraw all of the solution from the vial.
o Remove the needle of the syringe and attach a single use MiniSart filter unit.
o Use the plunger to filter out the ampicillin solution into 1.5 ml epp. tubes
Plating on Ampicillin Plates
o The transformed cells are poured onto LB Agar plates containing Ampicillin.
o Spreading is done by introducing 2-3 glass beads into the plate and then shaking vigorously in a lateral direction.
o The plates are incubated at 37oC for 24hrs.
Materials:
LB broth bacteria culture medium. The content are 1% Tryptone, 0.5% yeast extract, 200 mM NaCl, then it sterilize by autoclaving in suitable aliquots.
STET mix which is contain 5% (v/v) Triton X-I 00, 50 mM Tris-HCl, pH 8.0, 50 mM EDTA, pH 8.0, 8% (w/v) sucrose. Store the mix solution at room temperature.
Lysozyme: Dry powder. Store at -20oC.
70% Ethanol.
Isopropanol.
TE solution: 10 mM Tris-HCl pH 8.0,1 mM EDTA.
A boiling water bath: An opened bottom tube rack is required because the tubes must be placed directly in the water to achieve rapid heating.
Sterile wooden toothpicks.
Methods:
1. Set up a culture for each miniprep by inoculating 2-3 mL of L-broth, containing an appropriate antibiotic (e.g., 100 micrograms/mL ampicillin) with a bacterial colony. Grow overnight at 37oC with vigorous shaking.
2. Where plasmids have a high copy number, the growth time may be reduced to approx 6 h.
3. Before starting the miniprep, begin boiling the water and make up a fresh solution of 1 mg/mL lysozyme in STET mix.
4. Fill a 1.5-mL labeled microfuge tube with an aliquot from each culture. Pellet the bacteria by centrifugation for 1 min at 12,000 g. Carefully aspirate off the supernatant using a drawn-out Pasteur pipet.
5. The short centrifugation time leaves a loose pellet that is easier to resuspend. If the pellet does not readily resuspend, pipet the solution up and down to dislodge it. Do not suck the pellet directly into the pipet tip.
6. Vortex each pellet for a few seconds to break up the pellet. Add 20 micrliters STET mix to each tube. The pellet should now easily resuspend by vortexing.
7. Immediately place the tubes in the open-bottom rack, and place in the boiling water for exactly 45s. Ensure that each tube is at least half submerged.
8. Centrifuge the tubes at 12,000 g for 10 min. A large, sticky, loose pellet should form.
9. Remove the pellet from each tube by “fishing” it out with a sterile wooden toothpick. Because the pellet is quite slippery, it is useful to have a paper tissue at the top of the tube to catch the pellet and prevent it from slipping back down into the tube.
10. Add 200 microliters isopropanol to each tube, and centrifuge at 12,000 g for 5 min.
11. Aspirate the supernatant, and wash the pellet in 500 microliters 70% ethanol. Centrifuge the tube for 1 min to compact the pellet, and then aspirate the 70% ethanol.
12. Air dry the pellets for 10 min, and resuspend each one in 100 microliters TE buffer. Vortex and shake for 10 min before use to ensure complete dissolution.
13. Use 10 microliters (equivalent to 100 ng of plasmid for most vectors) and analyze by gel electrophoresis.
Colony PCR
Use a sterile toothpick or pipette tip to re-suspend a plated colony in 50μl sterile water.
Reaction Mix
1 μl 10x polymerase buffer
1 μl 10x dNTPs (10x = 2.5 mM each dNTP)
0.15 μl 40 μM FWD primer
0.15 μl 40 μM REV primer
0.1 μl Polymerase
6.6 μl H2O
1.0 μl template suspension
PCR protocol
95 C for 6 minutes (disrupt cells, separate DNA)
Cycle 35 times:
95oC for 30 s (melting)
53oC for 30 s (annealing)
72oC for 45 s (elongation)
72oC for 10 minutes (final elongation)
4oC for storage.
Isolation of bacterial plasmid
Materials:
1. TENS solution:
* 10 mM Tris (pH to 7.5)
* 1 mM ethylenediaminetetraacetic acid (pH to 8.0 to dissolve)
* 0.1 N sodium hydroxide
* 0.5 % sodium dodecyl sulfate
2. 3 M Sodium acetate, pH 5.2
3. Pre-chilled (at -20 degrees C) 100 % ethanol
4. 70 % Ethanol
5. Distilled water
6. Overnight bacterial culture
Supplies:
1. Micropipetter and tips
2. Vortex mixer
3. Microcentrifuge and tubes
Procedures:
1. Spin 1.5 ml of overnight bacterial culture for 30-60 seconds in a microcentrifuge.
2. Decant supernatant, leaving 50-100 ul in the tube.
3. Vortex to resuspend the bacteria pellet completely.
4. Add 300 ul of TENS solution.
5. Vortex for 5 seconds to mix.
6. Add 150 ul of the sodium acetate.
7. Vortex for 5 seconds to mix.
8. Spin for 2 minutes in a microcentrifuge.
10. Add 0.9 ml of pre-chilled 100 % ethanol.
11. Spin for 5 minutes in a microcentrifuge.
12. Discard supernatant and add 1 ml of 70 % ethanol.
13. Discard the ethanol and add another 1 ml of 70 % ethanol.
14. Withdraw and discard as much liquid (ethanol) as possible then air-dry the pellet.
15. Resuspend the pellet in 30 ul of distilled water and keep at 4 degrees C or -20 degrees C.
Precautions: DNA should be stored at -20 degrees C, but repeated freeze-thaw should be avoided. So if it is needed frequently or in a couple of days, store it at 4 degrees C.
DNA Kit Plate Instructions
To use the DNA in the Distribution Kit you may follow these instructions:
1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells.
2. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 1 or 2uL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.
- To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.
DNA Kit Plate Orientation
Top view of plates containing dry DNA; red circle indicates well 13H
The foil covers on each 384 well kit plate can be easily punched through with a pipette tip into the well of your choice. Unfortunately, the foil cover will also obscure both column and well markings. You can still find your part by correctly orienting the plate using the two notched corners as markers: well A1 is located at the upper left corner of the plate when the long side of the plate with the notched corners is considered the bottom.
Once you have found the well which your BioBrick™ part of choice is located in by searching for it through the Registry, you will want to count across the plate starting with Column 1 until you get to Column 13 and down the plate starting with Row A until you get to Row H.
Make sure that the two notched corners of the plate are oriented at the BOTTOM of the plate (see the "top view" image on the right for correct orientation)
Linearized Plasmid Backbone Usage
In addition to the DNA plates the Distribution Kit also contains a set of four linearized plasmid backbones: pSB1A3, pSB1C3, pSB1K3, and pSB1T3.
These plasmid backbones have been prepared via PCR and purified. Prior to ligation the linearized backbones will need to be digested with with EcoRI, PstI and DpnI, leaving two ends ready to be ligated to a Biobrick™ part.
All 2011 submissions will need to be in pSB1C3, so we recommend using our pSB1C3 backbone for that purpose. You can find more in depth instructions on how to use and make your own linearized plasmid backbones on the protocol page.
Note: The linearized plasmid backbones need to be cut by EcoRI and PstI restriction enzymes prior to use.
Reaction Mixtures
Amplification
o 1µl of sample DNA template
o 2µl of 10X buffer
o 1.2 µl of MgCl2
o 2µl of dNTP’s
o 0.5µl of KOD DNA polymerase
o 2.0µl of forward and reverse primers each.
o 9.3µl of water to bring the reaction volume to 20µl
Restriction Digest
Component Volume Water, nuclease-free 15 µl 10X FastDigest® buffer 2 µl DNA 2 µl (up to 1 µg) FastDigest® enzyme 2 µl Total volume 21 µl Ligation of Vector and Insert
Reaction Mix
o 3µl of Vector DNA
o 5µl of Insert DNA
o 1µl of Ligase
o 7µl of water
Colony PCR
0.1 μl Polymerase
6.6 μl H2O
1.0 μl template suspension
Plasmid isolation
1. TENS solution:
* 10 mM Tris (pH to 7.5)
* 1 mM ethylenediaminetetraacetic acid [EDTA] (pH to 8.0 to dissolve)
* 0.1 N sodium hydroxide
* 0.5 % sodium dodecyl sulfate[SDS]
2. 3 M Sodium acetate, pH 5.2.
3. Pre-chilled (at -20 degrees C) 100 % ethanol.
4. 70 % Ethanol.
5. Distilled water.
6. Overnight bacterial culture.
Gel Elution
7. Agarose Gel (low melt)
8. 100ml Tris-EDTA (T.E)
• 10mM Tris-HCl pH 7.6
• 1mM EDTA
9. 100ml Isopropanol/ phenol
10. 400ul Chloroform
11. 2.5ml Ethanol
12. Sodium acetate
Chemical competent cell preparation
5. 150ml Culture media
6. 150ml LB Broth
7. CaCl2 (25ml for 100ml start culture)
8. 2ml Glycerol
