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[IDT] 유전자 합성 후 plasmid transformation protocol [chemical]

Transformations (Chemical)

Place frozen competent cells and pre-labeled tubes on ice
Pre-warm the hot bath to 42°C
Once cells are thawed on ice, aliquot 25 µL of XL1 Blue cells to each of the pre-chilled tubes. Keep all the tubes on ice
Add 2 µL of ligation or PCR reaction to cells and gently swirl the cells with pipette tip
Incubate on ice for 30 minutes
Heat shock tubes for 45 seconds in 42°C hot bath
Return tubes to ice for 2 minutes
Add 250 µL of SOC Media to each tube and place tubes in a 37°C shaking incubator for 1 hour
Pre-warm agar plates to 37°C in incubator
Spread 125 µL of cells on agar plates
After spreading cells, let plates sit for approximately 10 minutes at room temperature. Then place the plates upside down in 37° incubator for 12 – 24 hours

Inoculation

For a large number of colonies, use a 2.2 mL deep well plate and add 1.6 mL of LB Broth with the appropriate antibiotic.
For a small number of colonies, use a 14 mL round bottom culture tube and add 2 mL of LB Broth with the appropriate antibiotic
Touch the colony with a toothpick or pipette tip and place it in the broth
Cover with a gas permeable lid and place in a 37° shaking incubator for 12 – 20 hours.

Digestions

The standard in-house digestion protocol is below:

Total Volume	50µL
DNA	400ng
Buffer	5µL
Water	XµL
Enzyme(s)	1µL each

Incubate for 1 hour at the temperature recommended by the enzyme manufacturer. Take 2-5 µL of the digested sample, add loading dye, and run on a gel to verify that the digestion took place. In separate lanes, also run a ladder to verify the size of the product and a sample of undigested product as a control.

*The length of running time for the gel will depend on the size of the digested insert. Smaller inserts require shorter run times or the product will run off the gel.

*The enzyme should not be more than 1/10^th of total reaction

Reagents

Material	Components		Amount (g or ml)/L
STE Buffer pH 7.5	10 mM	Tris	1.21
	10 mM	NaCl	2.92
	1 mM	EDTA	0.37
10X Taq Ligase Buffer pH 7.6	200 mM	Tris	24.22
	250 mM	Potassium Acetate	24.54
	100 mM	Magnesium Acetate (tetrahydrate)	21.45
	100 mM	DTT	15.42
	100 mM	NAD	6.85
	1 %	Triton-X100	24.22
1X T4 DNA Ligase Buffer pH 7.5	50 mM	Tris	6.05
	10 mM	Magnesium Acetate (tetrahydrate)	2.03
	10 mM	DTT	1.54
	1 mM	Adenosine 5′-triphosphate disodium salt	0.55
	25 µg/ml	BSA	0.025
Agar Plates with Amp	1 %	Tryptone	10
	1 %	Sodium Chloride	10
	0.5 %	Yeast Extract	5
	1.5 %	Agar	15
	100 µg/ml	Ampicillin	0.1
LB Broth with Amp	1 %	Tryptone	10
	1 %	Sodium Chloride	10
	0.5 %	Yeast Extract	5
	100 µg/ml	Ampicillin	0.1
Agar Plates with Kan	1 %	Tryptone	10
	1 %	Sodium Chloride	10
	0.5 %	Yeast Extract	5
	1.5 %	Agar	15
	50 µg/ml	Kanamycin	0.05
LB Broth with Kan	1 %	Tryptone	10
	1 %	Sodium Chloride	10
	0.5 %	Yeast Extract	5
	50 µg/ml	Kanamycin	0.05
SOC Media pH 7.5	2 %	Tryptone	20
	0.5 %	Yeast Extract	5
	0.05 %	Sodium Chloride	0.5
	2.5 mM	Potassium Chloride	0.19
	10 mM	Magnesium Chloride (hexahydrate)	20.3
1X Loading Dye	0.4 %	Bromophenol Blue	4
	0.4 %	Xylene Cyanol FF	4
	50 %	Glycerol	500