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Revision as of 13:31, 18 July 2011

Protocols

Resuspending DNA from registry-parts:

Poke a hole in foil of corresponding well
Resuspend DNA in 10 µL dH2O (pipette up and down a few times)
Wait 5 minutes.
Transfer the resuspended DNA to pCR tube and store in -20C.

Transforming competent cells:

Thaw competent cells on ice
Mix with 2 µL plasmid DNA
Incubate on ice 30 minutes
Heat-shock cells 45 seconds in 42C water-bath.
Incubate 5 minutes on ice
Add 200 µL SOC
Incubate the Eppendorf tubes in Erlenmeyer flask 37C shaking incubator for 2 hours
Plate 50 µl and 200 µl of the transformation onto the dishes, and spread.
Incubate ON in 37C

Isolating plasmids:

Inoculate one colony in 3 mL LB with appropriate antibiotic. (IE 1,5µL amp stock in 3 mL LB)
Grow in shaking incubator 30C ON
Isolate plasmid using [http://www.ebiotrade.com/buyf/productsf/Promega/tb225.pdf SV Miniprep procedure]
Store in -20C

Gel Extraction:

Using QIAquick Gel Extraction Kit [http://molecool.wustl.edu/krolllab/Kroll_Lab_Protocols/Molecular%20Biology%20protocols/Cloning%20protocols%20folder/Gel%20extraction-Qiagen.pdf protocol]
eluating with dH20

Restriction Digest:

Add 500ng of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.
Add 5ul of NEBuffer 2 to the tube.
Add 0.5µl of BSA to the tube.
Add 1µl of your first enzyme.
Add 1µl of your second enzyme.
There should be a total volume of 50ul. Mix well and spin down.
Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.

Ligation:

After digestion, separation, purification.
Add 11ul of dH20
Add 2ul from each sample you will be ligating (destination plasmid, and part)
Add 2ul of T4 DNA Ligase Reaction Buffer
Add 1ul of T4 DNA Ligase
Mix well, and spin down.
Incubate for 30min at 16C and 20min at 80C to heat kill.
Use 2ul of ligation to transform into competent cells.

Alternatively:

After gel extraction:
Add 1 µL T4 DNA Ligase Reaction Buffer
ca 6:1 molar ratio of insert to vector (~10ng vector)
Add (8.5 - vector and insert volume)μl
0.5 μL T4 Ligase

Testing of construct

Test 1 Nullmedium:

Inoculate in 5 mL ON
Dillute 4 x 1:100 in LB, grow to OD 0,5
Pellet, resuspend 2x in 0,9% NaCl, 37C, 2x in LB
Grow 3 hrs?

Test 2 Amino acid deprived:

Inoculate in 5 mL ON
Dillute 4 x 1:100 in LB, grow to OD 0,5
Pellet, resuspend 2x in no amino acid media, 37C, 2x in LB
Grow 3 hrs?

Test 3 Temperature:

Inoculate in 5 mL ON
Dillute 6 x 1:100 in LB, grow to OD 0,5
Pellet, resuspend in LB, Incubate 2 x 25C, 2 x 37C, 2x 40C ?
Grow 3 hrs?

Recipes used in the lab

LB Broth

Bacto-tryptone: 10 g/L
Yeast extract: 5 g/L
NaCl 5 g/L

Adjust pH to 7,4 with NaOH, autoclave.

LA

Dissolve LB
Add agar, 15 g/L

Adjust pH to 7,4 with NaOH, autoclave.

SOC:

Bacto-tryptone: 20 g/L
Yeast extract: 5 g/L
NaCl 0,5 g/L
10 mL 250 mM KCl (25mM) → 0,186 g / 10mL H20
5 mL 2M MgCl2 (10mM) → 0,952 g / 5mL

Autoclave, cool, then add:

20 mL 1M sterile-filtered glucose (20mM)

Ampicillin-plates:

Use stock 200 mg/mL. → 100µg/mL in liquid agar. I.E. 250 µL stock in 500 mL agar. Add when cool enough to pour.

Spectinomycin-plates:

Use stock 100 mg/mL. → 100µg/mL in liquid agar. I.E. 500 µL stock in 500 mL agar. Add when cool enough to pour.

Ampicillin-IPTG plates:

Ampicillin 100 µg / mL (500 µL amp stock (200 mg/mL) in 500 mL agar) IPTG 0,1 mM ( 63,5 µL IPTG stock (0,8 M))

M9 minimal medium:

0.4% carbon source, Glucose: 40 g
2mM MgSO4 → 0,2407 g / L 120,37
0.1mM CaCl2 → 0,0111 g / L
12,8g Na2HPO4.7H2O
3 g KH2PO4
0,5 g NaCl
1 g NH4Cl

In sterile H2O → 1000 mL

pH 7,4 , 4 x 250mL erlenmeyer, autoclave

@@ Line 65: / Line 65: @@
 *Add (8.5 - vector and insert volume)μl
 *0.5 μL T4 Ligase
 ==Testing of construct==

Team:NTNU Trondheim/Protocols