Team:BNDS CHINA/Experiments

Experiments

I. Primer design

Primers are designed by software Snapgene and Oligo 7. The following criteria are met when the most suitable primers are picked out.

1. the Tm value of overlapping with the template should be no more than 55°C;
2. a hairpin with Tm value less than the annealing temperature could be included;
3. the absolute value of ΔG of the most stable 3’-dimer is less than 7 kcal/mol.

II. PCR

Ingredients are mixed as follow:

Q5 Hot Start High-Fidelity 2X Master Mix System

INGREDIENTS	25 µl REACTION	50 µl REACTION	FINAL CONCENTRATION
Forward Primer	1.25 µl	2.5 µl	0.5 µM
Reverse Primer	1.25 µl	2.5 µl	0.5 µM
Template DNA	Variable	Variable	1,000 ng or less
Q5 Hot Start High-Fidelity 2X Master Mix	12.5 µl	25 µl	1X
Nuclease-Free Water	To 25 µl	To 50 µl

The program of PCR is set as:

Thermocycling Conditions for PCR

STEP	TEMPERATURE	TIME
Initial Denaturation	98°C	30 seconds
35 Cycles	98°C	10 seconds
	Tm-5°C	20 seconds
	72°C	20-30 seconds/kb
Final Extension	72°C	2 minutes
Hold	16°C	∞

III. DpnI digestion

1 µl DpnI is added into the PCR tube, then the PCR tube is placed into the 37°C incubator for 15-30 minutes.

IV. Gel electrophoresis

Prepare the gel solution by adding 1 g agarose in to100 ml TAE 1X buffer.

Heat until all agarose has dissolved completely.

Add 10µl Gelstain into the cooled solution (at around 60°C), mix the solution evenly through shaking.

Pour the solution into a cast and place the corresponding comb on top of the gel cast.

Wait for the gel to solidify (about 30 minutes).

Transfer the solid gel into the electrophoresis apparatus with TAE 1X buffer submerged.

Add 10 µl DNA samples and 5 µl TransGene DNA Ladder into cells of the gel.

Run the electrophoresis at 110 V for 30 minutes.

Visualize the gel under UV light.

V. DNA sequencing

When designing primers for sequencing, notice that sequencing primers are one-way with a Tm value at about 60-62°C. The starting point should be 100bp before the restriction site, and one primer can be responsible for sequencing 700 bp accurately. Repeat this process of 700 bp until another restriction site is reached. No additional nucleotides are required for sequencing.

VI. Gel Extraction

• Objective: Purify DNA from the gel after PCR or after migration

  	Volume
  Cut DNA band (with gel)	n (note that 100mg gel=100µl)
  GSB buffer	≦3n
  isopropanol	n
  Nuclease free water (dd H2O)	40 µl

  1. Weight the EP tube that is going to be used.
  2. Cut the bands, as small as possible, from the gel and put them into separate EP tube. (the gel should be placed under ultraviolet light)
  3. Weight the tube with gel again and calculate the mass for the gel
  4. Add ≦ 3 volume of GSB for 1volume of gel (100 mg gel = 100 µl).
  5. Heat with 55 °C until the gel is entirely dissolved. If the solution shows a color of purple, add sodium acetate (pH=5.2) until the solution shows the same color withe GSB (yellow).
  6. Add 1 volume of isopropanol in order to obtain higher concentration of DNA solution.
  7. Waite until the temperature of mixed solution drops to room temperature.
  8. Put the mix into spin column, centrifuge at 10,000× g (10,000 rpm) for 1min. Discard the effluent.
  9. Place the column into a clean EP tube.
  10. Add 650 µL WB buffer, centrifuge at 10,000× g (10,000 rpm) for 1min. Discard the effluent.
  11. Centrifuge again for 1-2 min in order to remove residual WB.
  12. Place the column into another clean EP tube and heat them with 65 °C heat bath (lid open), until the remaining ethanol evaporate completely. Heat the nuclease free water at the same time.
  13. To elute DNA add 40 µl nuclease free water to the center of the column and centrifuge for 1 min at 13000 rpm.
  14. Use 1 µL of the product for Nanodrop testing