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====== Protocols-Standard operating Procedures (SOP) ======

===== Plasmid DNA isolation_ alkaline lysis mini prep =====


Modified from: Sambrook & Russell 2001, Kirchhof 2002

Materials & Reagents
Alkaline lysis solution I: 50mM glucose, 25mM Tris-Cl pH 8.0, 10mM EDTA pH 8.0
Alkaline lysis solution II: 0.2N NaOH, 1% SDS; make fresh
Alkaline lysis solution III: 60ml 5M potassium acetate, 11.5ml glacial acetic acid, 28.5ml ddH2O; place on ice before use
PCI: 25:24:1 phenol (pH 8.0):chloroform:isoamyl alcohol; make fresh
Chloroform
Ethanol (100%, 70%)
3M Sodium acetate (pH 5.2)
TE buffer: 10mM Tris-Cl pH 8.0, 1mM EDTA pH 8.0

Protocol

1.	Inoculate culture in 10ml LB, incubate overnight at 37C at 200rpm.
2.	Place 1.5ml of culture in an Eppendorf tube, centrifuge 13,000xg for 5min. Discard supernatant.
3.	Resuspend cells in 100μl Alkaline lysis solution I
4.	Add 200μl of Alkaline lysis solution II; mix by inversion. Place tube on ice for 10 minutes
5.	Add 150μl of ice-cold Alkaline lysis solution III, vortex 10s. Place tube on ice for 10 minutes or until thick white precipitate is observed.
6.	Centrifuge lysate at 13,000xg for 15min. Transfer supernatant to fresh tube; discard pellet
7.	Add 1ml PCI to supernatant, vortex 30s
8.	Centrifuge 10min at 13,000xg
9.	Transfer upper aqueous layer to a new tube; be careful not to transfer any lower organic layer (discarded). 
10.	Add 1ml chloroform, vortex 30s
11.	Centrifuge 10min at 13,000xg
12.	Transfer upper aqueous layer to a new tube; be careful not to transfer any lower organic layer (discarded). 
13.	Add 1ml 100% ethanol and 50μl of 3M sodium acetate (pH 5.2), vortex 10s. Place on ice for at least 30min to precipitate plasmid.
14.	Centrifuge 10min at 30,000xg. Remove and discard supernatant.
15.	Rinse nucleic acid pellet with 1ml of 70% ethanol
16.	Centrifuge 10min at 13,000xg. Remove and discard supernatant.
17.	Resuspend pellet in 50μl of TE.
18.	Quantify DNA using a Spectrophotometer.

===== DNA quantification_ using Spectrophotometer =====


Materials & Reagents
UV-transparent cuvettes

Protocol
1.	Rinse UV-transparent cuvette with ddH2O
2.	Blank the spectrophotometer at 260nm with 1ml of ddH2O
3.	Add 999μl of ddH2O to the cuvette
4.	Add 1μl of DNA (dilute 1000x)
5.	Wrap cuvette opening with parafilm, invert to mix content
6.	Remove parafilm. Take absorption reading at 260nm
7.	Calculate DNA concentration using the following formula:
           1.0 absorbance unit at OD260 = 50μg/ml dsDNA


===== Plasmid (DNA) restriction_ with EcoRI =====

Modified from: Manufacturer (Fermentas) Recommendations 

Materials & Reagents
10u/μl EcoRI (Fermentas ER0271)
10x Buffer Tango (Fermentas #BY5)
37C water bath
65C water bath or heatblock

Protocol

1.	Mix reagents as follows:

ddH2O	               14.0 μl
10x Buffer Tango	4.0 μl
DNA (0.5-1μg/μl)	1.0 μl
10u/μl EcoRI	        1.0 μl
Total	               20.0 μl

(final concentration = 2x)
(final concentration = 0.5u/μl)

2.	Mix gently and spin down for a few seconds
3.	Incubate at 37C for 1h
4.	Inactivate enzyme by incubating at 65C for 20min

===== Polymerase Chain Reaction (PCR) =====


Materials & Reagents
10x PCR buffer (BioLabs #B9004S)
10mM dNTP (BioLabs #N0447S)
Forward primer
Reverse primer
1u/μl Taq (BioLabs #M0267S)
Agarose 
1x TAE buffer (40mM Tris-acetate, 1mM EDTA)
1μg/ml Ethiodium bromide
2x loading dye (40% sucrose, with bromophenol blue)
1kb ladder (Fermentas #SM0313)


Protocol

1.	Mix reagents in each PCR tube as follows:

ddH2O	                       13.0 μl	
10x PCR buffer	                2.0 μl	
10mM dNTP	                1.0 μl	
Forward primer	                1.0 μl	
Reverse primer	                1.0 μl	
1u/μl Taq	                1.0 μl	
DNA template (0.5-1μg/μl)	1.0 μl	Include a negative control without template – add 1μl ddH2O
Total	                       20.0 μl	

2.	Set up and run PCR as follows:

Step 	Temperature 	Time 
2.1		95C	2 min
2.2		95C	30s
2.3		Annealing temperature	30s
2.4		72C	Extension time
2.5	 	Repeat steps 2.2-2.4 	30 times
2.6		72C	10 min
2.7		4C	hold

3.	Inspect PCR product by agarose gel electrophoresis:

3.1	Follow instructions to set up gel unit
3.2	Make 1% agarose gel: 0.5g agarose in 50ml TAE, microwave ~50s or until dissolved
3.3	Sit on bench ~10min to cool
3.4	Add 5μl ethidium bromide, mix well, pour; set comb
3.5	Solidify for at least 30min
3.6	When loading: mix 3μl PCR product with 3μl 2x loading dye on parafilm
3.7	Samples run towards cathode (red); run at 100V for 30 min before imaging under UV.
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