Team:HZAU-China/WetLab/Protocol
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Protocol
Overview:
1、 Get parts from kit
2、 Translation
3、 Pick single clone and enlargement culturing
4、 The extraction of plasmid
5、 Enzyme digestion、 purification and connection
6、 Check (PCR、Restriction Maps 、sequencing)
1、Get parts from kit
To use the DNA in the Distribution Kit, follow these instructions:
Note: There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/ul.
1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. 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. Transform 1ul of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
3. Pick a single colony and inocubate broth (again, with the correct antibiotic) and grow for 16 hours.
4. Use the resulting culture to miniprep the DNA and make your own glycerol stock.
* 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.
Note: There is not enough DNA in each well to perform anything but transformations
2、 Transformation
1)ECOSDH5@ :
2)Commom DH5@:
Standard heat-shock transformation of chemically competent bacteria.
1.Take competent cells out of -80°C and thaw on ice (approximately 20-30min).
2.Take agar plates (containing the appropriate antibiotic) out of 4°C to warm up to room temperature or place in 37°C incubator.
3.Mix 1 to 5μl of DNA (usually 10pg to 100ng) into 20-50μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
4.Place the competent cell/DNA mixture on ice for 20-30min.
5.Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 60~90 seconds (75sec is usually ideal).
Put the tubes back on ice for 2 min.
Add 250-500μl LB or SOC media (without antibiotic) and grow in 37°C shaking incubator for 45min.
Plate some or all of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.
Incubate plates at 37°C overnight.
3、 Pick single clone and enlargement culturing.
1.When ready to grow culture, add liquid LB to a tube or flask and add the appropriate antibiotic to the correct concentration.
2.Using a sterile pipette tip , select a single colony from LB agar plate.
Drop the tip or toothpick into the liquid LB + antibiotic and swirl.
Loosely cover the culture with sterile aluminum foil or a cap that is not air tight.
3.Incubate bacterial culture at 37°C for 12-18hr in a shacking incubator.
After incubation, check for growth, which is characterized by a cloudy haze in the media (see right).
Note: A good negative control is LB media + antibiotic without any bacteria inoculated. You should see no growth in this culture after overnight incubation.
4.For long term storage of the bacteria, we proceed with Creating a Glycerol Stock.
5.Then isolate the plasmid DNA from the bacterial culture by following the extraction of plamid.
4、 The extraction of plasmid
General Procedure-Genemark Plasmid Miniprep Purification Kit II
1. Pellet 1~10 ml of bacteria culture by centrifugation for 1 min at top speed (12~14,000x g) in a microcentrifuge. Discard the supernatant and remove any excess media.
Note: For liquid culture > 5 ml, increase Solution I, Solution II and Solution III volume to prevent product loss.
2. Resuspend the cell pellet completely in 200 μl of Solution I by pipetting or vortexing.
3. Add 200 μl Solution II and mix by inverting the tube 5 times. The cell suspension should turn clear immediately.
4. Add 200 μl Solution III and mix by inverting the tube 5 times.
5. Centrifuge the lysate at top speed in a microcentrifuge for 5 min. A compact white pellet will form along the side or at the bottom of the tube.
6. Insert the Spin Column into a Collection Tube, carefully transfer all of the clear lysate from step 5 to spin column, centrifuge at top speed for 1 min.
7. Discard the filtrate in the collection tube and add 500 μl Wash Solution A to the spin column and centrifuge for 1 min.
* This step can greatly reduce endonuclease contamination of EndA+ (e.g. BL21, DE3, HB101) and JM series (e.g. JM109) strains.
8. Discard the filtrate from the collection tube and add 700 μl of Wash Solution B to the spin column and centrifuge at top speed for 1 min. Repeat this step once more.
9. Discard the filtrate and centrifuge at top speed for additional 3~5 min to remove residual trace of ethanol.
* If centrifugation speed is lower than 10,000 g or residual ethanol must be removed completely, incubate the spin column in a heat oven (45~60°C) for 5 min to evaporate all of the ethanol.
10. Transfer the spin column into a new microcentrifuge tube and add 50~100 μl of Elution Solution or H2O (pH 7.0~8.5) into the column and wait for 1~2 min.
(For plasmid DNA larger than 7 kb, use preheated (60~70°C) Elution solution to elute.)
11. Centrifuge at top speed for 1 min to elute the DNA. Store the eluted plasmid DNA at -20°C. *The yield of plasmid DNA is 6~60 μg for 1~10 ml E. coli culture at purity of 1.8~2.0 (A260/A280).
5、 Enzyme digestion、 purification and ligation
Protocol for double digestion
For purify the digestion product:(50 system)
Pipette the following into a 1.5ml microfuge tube:
Enzyme A 2μl
Enzyme B 2μl
10×buffer 5μl
DNA 0.5-1μg
ddO H2 rest of the volume
incubate at recommended temperature (37℃) for at least30mins.
Purify the digestion product;
Notes: The enzymes used here are TaKaRa quick cute enzymes(EcoRI/XbaI/ SpeI/ PstI), and bufferM or His suitable for most of double digestion;
AxyPrep DNA Gel Extraction Kit:
For the rapid purification of DNA fragments from agarose gels
1. Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove residual buffer. Transfer the gel slice to a piece or plastic wrap or a weighing boat. Mince the gel into small pieces and weigh. In this application, the weight of gel is regarded as equivalent to the volume. For example, 100 mg of gel is equivalent to a 100 μl volume. Transfer the gel slice into a 1.5 ml microfuge tube.
Note: Alternatively, the gel slice can be placed into the 1.5 ml microfuge tube and then crushed with a pipette tip or other suitable device. Spin the tube for 30 sec at 12,000xg to consolidate the gel at the bottom of the tube.
Use the graduations to estimate the volume of the agarose gel
2. Add a 3x sample volume of Buffer DE-A.
Note: The color of Buffer DE-A is red. This color is used to add contrast in the next step, so that any pieces of unsolubilized agarose can be visualized.
3. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75°C until the gel is completely dissolved (typically, 6-8 minutes). Heat at 40°C if low-melt agarose gel is used. Intermittent vortexing (every 2-3 minutes) will accelerate gel solubilization.
IMPORTANT: Gel must be completely dissolved or the DNA fragment recovery will be reduced.
IMPORTANT: Do not heat the gel for longer than 10 minutes.
4. Add 0.5x Buffer DE-A volume of Buffer DE-B, mix. If the DNA fragment is less than 400 bp, supplement further with a 1x sample volume of isopropanol.
Example: For a 1% gel slice equivalent to 100 μl, add the following:
• 300 μl Buffer DE-A
• 150 μl Buffer DE-B
If the DNA fragment is <400 bp, you would also add:
• 100 μl of isopropanol.
Note: The color of the mixture will turn yellow after the addition of Buffer DE-B. Please make sure the contents are a uniform yellow color before proceeding.
5. Place a Miniprep column into a 2 ml microfuge tube (provided). Transfer the solubilized agarose from Step 4 into the column. Centrifuge at 12,000xg for 1 minute.
6. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 500 μl of Buffer W1. Centrifuge at 12,000xg for 30 seconds.
7. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 700 μl of Buffer W2. Centrifuge at 12,000xg for 30 seconds.
Note: Make sure that 95-100% ethanol has been added into Buffer W2 concentrate. Make a notation on the bottle label for future reference.
8. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Add a second 700 μl aliquot of Buffer W2 and centrifuge at 12,000xg for 1 minute.
Note: Two washes with Buffer W2 are used to ensure the complete removal of salt, eliminating potential problems in subsequent enzymatic reactions, such as ligation and sequencing reaction.
9. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Centrifuge at 12,000xg for 1 minute.
10. Transfer the Miniprep column into a clean 1.5 ml microfuge tube (provided). To elute the DNA, add 25-30 μl of Eluent or deionized water to the center of the membrane. Let it stand for 1 minute at room temperature. Centrifuge at 12,000xg for 1 minute.
Note: Pre-warming the Eluent at 65°C will generally improve elution efficiency.
Note: Deionized water can also be used to elute the DNA fragments.
LIgation:
DNA Ligation Kit Ver.2.1
Protocol for ligation (10μl system)
Pipet the following into a 0.5ml microfuge tube:
Insert DNA 3:1 molar excess over vector
Linearized vector DNA around 100ng
T4 DNA Ligase(TaKaRa) 1μl
10xT4 DNA Ligase 1μl
Buffer(TaKaRa)
ddO H2 rest of the volume
Vortex thoroughly and spin briefly;
Incubate the mixture at 16℃ at PCR machine or incubater about 1 h.
6、 Check(PCR、Restriction Maps 、sequencing)
1.PCR verufication (clony PCR) (10ul system)
2.For Enzyme digest verification:(10ul system)
Pipette the following into a PCR tube:
Enzyme A 1μl
Enzyme B 1μl
10×buffer 1μl
DNA 0.1-0.2μg
ddO H2 volume
incubate at recommended temperature (37℃) for at least30mins.
3.send the plasmid sample to company to sequencing.
Reference:
© 2015 Huazhong Agricultural University iGEM Team. All rights reserved.
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