Last Updated: 02/17/16; 2:16 AM
General Molecular Biology Protocols
Transformed Cells:
Store cells in 15% (or greater) Glycerol at –80°C.
Store cells in 3.5% DMSO at –80°C.
Pouring LB plates:
1.
LB with agarose is made up in approx 1L and autoclaved with stir bar
(approximately 30 minutes).
2.
Flask is then placed in 50°C waterbath so that Ampicilian (concentration
???) can be added.
3.
Pour LB/Agarose into plate so that the bottom is covered. Stack poured
plates in groups of 10 to decrease condensation as they cool.
4.
Allow to cool overnight at room temperature.
5.
Plates are marked with a black stripe to indicate Amp resistance.
Transformation:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Thaw competent cells (50l volume)
Place AMP+plate at 37°C
Add 10 ul of plasmid to cell tube
Incubate tube on ice for approx 30 min
Heat shock cells (37° or 42°C) for 20 sec
Place cells on ice for a few min
Bring volume up to 1ml with LB media
Shake at 37°C for 30 min to 1 hour
Spin cells down for 30 sec
Remove approx 900 ul (disgard)
Plate remaining volume and grow overnight 37°C
Pick colonies and culture for miniprep
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Last Updated: 02/17/16; 2:16 AM
Plasmid 10 Minute Miniprep
Solutions:
1)
2)
3)
TENS (make fresh each time):
To 4.5 ml TE add:
250 ul 10% SDS
250 ul 2N NaOH
Sodium Acetate 3.0M; pH 5.5
TE with 10ug/ml of RNAse A
Procedure:
1.
Spin 1.5 ml of overnight culture for 30 sec in microfuge
2.
Aspirate off all but 100ul of the supernatant and resuspend the pellet by
vortexting.
3.
Add 300ul of TENS and mix by inversion. The solution should become
viscous.
4.
Add 150ul of sodium acetate and vortex. A fine white precipitate should
form.
5.
Centrifuge for 2.5 minutes at 10K.
6.
TRANSFER the supernatant to a clean tube and add 2 volumes (1ml) of
room temperature EtOH.
7.
Vortex and pellet DNA by centrifugation for 2-5 minutes at 10K.
8.
Wash pellet with 70% ethanol and allow the pellet to dry.
9.
Resuspend the pellet in 30ul of TE with RNAseA.
10.
Digest 5-10ul as usual.
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Last Updated: 02/17/16; 2:16 AM
Example Digestion Reaction – 20 l reaction volume
Buffer (10X)
Restriction Enzyme
DNA
dH2O
2 ul
1 ul
6 ul
11 ul
if BSA is required by enzyme, reduce amount of water added.
0.7% Agarose Gel (Buttrick Lab)
for 300 ml:
2.1 grams of SeaKem Agarose
300 ml of 1X TAE
21 ul of EthBr (add once agarose is cooled to < 60°C)
Maxiprep
Grow up 500 ml culture with AMP in a 2000 ml flask overnight. Prepare via Qiagen kit.
Check purity and concentration of DNA by absorbance at 240, 260, 280. Purity is
determined by A260/A280 and should be above 1.5. Concentration is determined by the
following:
A260 * 50 * dilution / 1000 = X ug/ul.
Gel Purification of DNA fragments
Run approximately 2-4 ug of DNA on 0.7% gel to isolate desired fragment.
Ligation Reaction (eg. preferably use 10 ul reaction volume)
Ligation Ligation +
T4 DNA Ligase
1 ul
T4 DNA Ligase
Ligase buffer (5X)
2 ul
Ligase buffer (5X)
Vector
2 ul
Vector
dH2O
5 ul
Insert
Incubate 1 hour at room temp and then at 14°C overnight
use approximately 3:1 of insert to vector for ligation.
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1 ul
2 ul
2 ul
5 ul