Team:SJTU-BioX-Shanghai/Protocol

Protocol

PCR

Materials:

Buffer
Mg²⁺
dNTP
Template
F/R-primer
DNA enzyme

Methods:

1.Combination of all reagents

Name of reagent	volume (μl)
Buffer	2
Mg²⁺	2
Template	20 ng for plasmid or 200 ng for genome
F/R-primer	0.6 for each
Enzyme	0.4
Sterile Water	make reaction up to 20μ in total

2.Set the PCR machine to run the following steps:

Stage	Temperature (℃)	Time
initial denaturation	94	2 min
	94 (denaturation)	15 seconds
30 cycles	40-55 (annealing)	30 seconds
	68 (extension)	30 seconds per bp
Final extension	68	7 min
Incubate	12	Infinite

Restriction Digestion

Materials:

Restriction Enzyme: Fisher FastDigest Restriction Enzyme
Green Buffer
DNA samplesr
Sterile Water

Methods:

Note: the components listed below are for double digestion!

Component	volume (μl)
Green Buffer	2
DNA sample	600 ng for plasmid or up to 200 ng for PCR production
Template	20 ng for plasmid or 200 ng for genome
Restriction Enzyme	1 for each
Sterile Water	make reaction up to 20μ in total

Set up the reaction following the above table and incubate at 37℃ for 15-30 min and do the gel electrophoresis or purification later

Ligation

Materials:

T4 DNA Ligase
T4 DNA Ligase Buffer
Vector Plasmid
Insert DNA
Sterile Water

Methods:

Component	volume or mass
T4 DNA Ligase Buffer	2 μ
Vector Plasmid	50 ng
Insert DNA	require molar ratio of 3:1 to 7:1 (insert:vector)
T4 DNA Ligase	0.2 μ
Sterile Water	make reaction up to 20μ in total

Make the reaction and incubate at room temperature for 4-6 hours

Note: for how to calculate volumes of vector and insert DNA, we highly recommend NEBbioCalculator

Sterile Water

DNA Gel Electrophoresis

Materials:

Agarose
TBE Buffer
Gel board
EB
DNA×10 or×6 Loading Buffer
Electrophoresis tank
sample
marker 2000 or 15000

Methods:

1. Make a 1.0%-1.5% Agarose gel according to the volume of your gel board (eg. add 0.45g Agarose in 30ml TBE if choose a concentration of 1.5%)

2. Heat the container with the mixture until it is complete dissolved. When the solution starts boiling, stop heating, shake the container until materials mix well and heat again until the mixture become clear

3. Wait until the solution cools down to 50℃,add 0.01% EB (eg. 3μin 30ml) or non-poisonous dyes instead

4. Pour the solution into a gel board and insert the comb

5. Set the gel at room temperature and wait until it completely solidifies

6. Remove the comb and transfer the gel to the electrophoresis tank

7. Add samples into pores with 40% DNA Loading Buffer (eg. 2μLoading Buffer in 5μDNA sample)

8. Run the gel for 20 min at 100V, 100mA

Transformation

Materials:

LB broth
Ice
Selection plates with corresponding antibiotics.

Purification and Gel Extraction & Plasmid Extraction

Cotransformation

Characterization