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Revision as of 09:27, 2 August 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

PCR

PCR mix:

Work on ice
0.5 µL DNA
5 µL 10x PCR buffer 2 (w/MgCl₂)
0.5 µL 10 mM dNTPs
1 µL fwd primer
1 µL rev primer
0.5 µL polymerase
41.5 µL H20

PCR program:

Temperature of lid: 103°C
Heat cycles:
Initial denaturation: 94°C for 2 min
Denaturing: 94°C for 30 s
Annealing: 58°C for 30 s
Elongation: 72°C for 60 s
Repeat 2-4 34 times
Final elongation: 72C 7 min
Cooling: 4 C HOLD

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?

TSS competent cells preparation:

Make TSS
Inoculate 5 ml LB (w/o antibiotic) from company stock stored in –80C and let grow o/n.
Inoculate 30 ml LB (w/o antibiotic) with 300 μL from the o/n culture. (Alternative: Inoculate 3 ml for 5 hrs and dump into 30ml culture.)
Monitor OD until it reaches 0.4-0.5.
When OD reaches 0.4 remove from shaker and put on ice.
Pellet the cells in 50ml falcon (or autoclaved tube) tube at 6000g, 4C for 10 minutes.
Decant liquid and place cells back on ice.
Resuspend cells in 3.0ml TSS, pipet up and down and vortex. (resuspend in 10% original volume of TSS. ie. 30ml culture, 3 ml TSS)
Pipet 250 μL cells into each prelabeled and precooled microfuge tube
Store at –80C

Making blunt ends on DNA with sticky ends:

T4 DNA polymerase was used (Fills in 3' overhang and removes 5' overhang.

Digested DNA was cooled on ice, 2 µl 2 mM dNTP and 1 µl T4-DNA polymerase was added.
The mix was incubated at 16 C for 12 min.
Inactivate enzyme and isolate fragment by using PCR purification kit.

Recipes used in the lab

LB medium

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

Make M9 salts

To make M9 Salts aliquot 800ml H2O and add
- 33.93g Na2HPO4 16,7 g
- 15g KH2PO4 7,5g
- 2.5g NaCl 1,25g
- 5.0g NH4Cl 2,5g
- Stir until dissolved
- Adjust to 1000ml with distilled H2O
- Sterilize by autoclaving
Measure ~700ml of distilled H2O (sterile)
Add 200ml of M9 salts
Add 2ml of 1M MgSO4 (sterile)
Add 20 ml of 20% glucose (or other carbon source)
Add 100ul of 1M CaCl2 (sterile)
Adjust to 1000ml with distilled H2O

Transfection Storage Solution (TSS)

50 ml TSS:

10 % Polyethylene glycol 5 g
5 % Dimethyl Sulfoxide (DMSO) 2,5 ml
20 mM MgCl2 1 ml
Bring to 50 ml with autoclaved LB
Stir until dissolved
Filter through 0.22 μM syringe filter
Autoclave centrifuge tubes, flasks and bottle/beaker for TSS
Store at -20 C

@@ Line 132: / Line 132: @@
 = Recipes used in the lab =
-== LB Broth ==
+== LB medium ==
 *Bacto-tryptone:	10 g/L

Team:NTNU Trondheim/Protocols