Team:NTHU Taiwan/Protocol

Protocol

Cloning

PCR amplification

Normal PCR/ Gradient PCR (find best annealing temperature)

PCR mixture (YCL lab):

item	volume
YCL lab Phusion polymerase premix(dNTP 10mM, Phusion pol, ddH2O, 5X buffer)	47ul
DNA template	1ul
Primer forward 10uM	1ul
Primer reverse 10uM	1ul

DNA amount: 100ng
primer concentration: 10uM

PCR program:

temperature	Preheat
95&degC	5min
95&degC	30s
55&degC (gradient determined best temp.)	30s
72&degC	2-3min, go back to step 3 repeat for 40 cycles.
72&degC	10min
4&degC	o/n

PCR cleanup (with Geneaid Gel/PCR Kit (DFH100, DFH300))

I. Transfer reaction product to a 1.5 ml microcentrifuge tube. Add 5 volumes of Gel/PCR Buffer to the sample then vortex.(*If the mixture has turned from yellow to purple, add 10 µl of 3M sodium acetate (pH5.0) and mix thoroughly.)

II. Transfer the sample mixture to the DFH Column. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through.

III. Add 600 µl of Wash Buffer (make sure absolute ethanol was added) into the DFH Column and let stand for 1 minute. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through. Centrifuge again at 14-16,000 x g for 3 minutes to dry the column matrix.

IV. Transfer the dried DFH Column to a new 1.5 ml microcentrifuge tube. Add 20-50 µl of water into the CENTER of the column matrix. Let stand for at least 2 minutes to allow water to be completely absorbed. Centrifuge at 14-16,000 x g for 2 minutes at room temperature to elute the purified DNA.

V. If the final concentration of purified DNA is too low (lower than 15ng/ul), we would put the elution buffer (in our protocol is ddH2O in 55°c) with purified DNA back to the column and stand for 10 minutes to elute the remained DNA.

Digestion (10ul reaction)

I. Set up the following reaction in a microcentrifuge tube on ice. (10ul reaction)

Digest for 1 to 2 hours
Use NED double digest finder https://nebcloner.neb.com/#!/redigest to find the best condition for digestion. EcoRI-HF, SpeI-HF, PstI and XbaI-HF are often used enzyme in iGEM.
Concentration of restriction enzyme should be lower than 5% to avoid star activity.

item	volume
DNA (depends on concentration)	1ug
Cut Smart buffer (10X)	2ul
Enzyme 1	0.3ul
Enzyme 2	0.3ul

II. Heat inactivate at 65°C / 80°C for 20 minutes.

Gel electrophoresis (Before ligation to determine the amount of DNA)

I. Making 1.3% agarose gel (small)

TAE buffer (1X) 25mL + Agarose 0.33g

II.Loading sample

DNA marker: 1ul of 6X (loading dye/Novel juice) + 5 ul of DNA ladder

DNA sample: 1.5ul of 6X (loading dye/Novel juice) + 2 ul of DNA

III.Start the electrophoresis 100V for 30 minutes.

Ligation

Method 1: Use Nanodrop to measure the concentration of DNA sample and determine the volume used to ligate.

Method 2: Use the result of gel electrophoresis. (brightness of DNA sample to determine the volume used to ligate.)

I. Set up the following reaction in a microcentrifuge tube on ice. (20ul reaction)

item	volume
T4 DNA ligase	0.5ul
T4 DNA ligase buffer (10X)	2ul
Insert sequence	The volume left in tube
Backbone	The volume of insert sequence/2orThe volume of insert sequence/3
ddH2O	To 20ul

II.Incubate at 16°C overnight.

III.Heat inactivate at 80°C for 20 minutes.

Transformation

I. Transformation

II. Add 1-5µl containing ligation product to the 100ul of the cells. (DNA cannot exceed 10% of the total volume of competent cells.)

III. Place the mixture on ice for 30 minutes.

IV. Heat shock at exactly 42°C for exactly 45 seconds.

V. Place on ice for 5 minutes.

VI. Do IV , V again.

VII. Pipette 900 µl of room temperature LB broth without antibiotic into the mixture. (for recovery)

VIII. Place at 37°C for 60 minutes.

IX. Warm selection plates to 37°C.

X. Centrifuge at 14,000-16,000xg to remove the excess LB and resuspend the cells with 200ul of LB with antibiotic. Spread onto a selection plate and incubate for 16 hours at 37°C.

Miniprep (with Geneaid Miniprep kit)

I. Harvesting

Transfer 1.0-1.5 ml of cultured bacterial cells to a microcentrifuge tube. Centrifuge at 14-16,000 x g for 1 minute at room temperature to form a cell pellet then discard the supernatant completely.

II. Resuspension

Add 200 µl of PD1 Buffer (make sure RNase A was added) to the 1.5 ml microcentrifuge tube containing the cell pellet. Resuspend the cell pellet completely by vortex or pipette until all traces of the cell pellet have been dissolved.

III. Cell Lysis

Add 200 µl of PD2 Buffer to the resuspended sample then mix gently by inverting the tube 10 times. Close PD2 Buffer bottle immediately after use to avoid CO2 acidification. Do not vortex to avoid shearing the genomic DNA. Let stand at room temperature for at least 2 minutes to ensure the lysate is homogeneous. Do not exceed 5 minutes.

IV. Neutralization

Add 300 µl of PD3 Buffer then mix immediately by inverting the tube 10 times. Do not vortex to avoid shearing the genomic DNA. Centrifuge at 14-16,000 x g for 3 minutes at room temperature. If using >5 ml of bacterial cells, centrifuge at 16-20,000 x g for 5-8 minutes. During centrifugation, place a PDH Column in a 2 ml Collection Tube.

V. DNA Binding

Transfer all of the supernatant to the PDH Column. Use a narrow pipette tip to ensure the supernatant is completely transferred without disrupting the white precipitate. Centrifuge at 14-16,000 x g for 30 seconds at room temperature then discard the flow-through. Place the PDH Column back in the 2 ml Collection Tube.

VI. Wash

Add 600 µl of Wash Buffer (make sure absolute ethanol was added) into the PDH Column. Centrifuge at 14-16,000 x g for 30 seconds at room temperature. Discard the flow through then place the PDH Column back in the 2 ml Collection Tube. Centrifuge at 14-16,000 x g for 3 minutes at room temperature to dry the column matrix. Transfer the dried PDH Column to a new 1.5 ml microcentrifuge tube. Perform Wash Buffer steps twice for salt sensitive downstream applications.

VII. Elution

Add 50 µl of water into the CENTER of the column matrix. Let stand for at least 2 minutes to allow Elution Buffer, TE or water to be completely absorbed. Centrifuge at 14-16,000 x g for 2 minutes at room temperature to elute the purified DNA.