Revision as of 14:19, 3 September 2008

Newcastle University

GOLD MEDAL WINNER 2008

Home	Team	Original Aims	Software	Modelling	Proof of Concept Brick	Wet Lab	Conclusions

Home >> Wet Lab >> Protocols

Agarose Gel Electrophoresis

Add 20ml 50 X TAE to 980ml H2O.
Add 3g agarose powder to 300ml of the TAE-H2O (1 x TAE) solution.
Microwave full power for 5 minutes or until solution is clear and agarose has dissolved.
Leave to stand for 5-10 minutes. Put tape around sides of tray.
Pour solution into tray, add 4μl of ethidium bromide and mix gently using the comb.
Clean the comb (this prevents capillary action drawing the gel up between the comb teeth and making 'spikes' around the wells) and reinsert near one end of the tray.
Leave to set 30-40 mins.
Remove tape and comb, place tray in electrophoresis machine and pour 1 x TAE solution to just cover the gel.
Combine 1μl of loading buffer for every 5μl sample being loaded and mix by pipetting up and down.
Load marker and samples and run the gel.

N.B. We have found that a thin gel works best for loading small samples, but a thicker gel may be preferable if lots of sample needs to be loaded (for example if a fragment needs to be cut from the gel). A comb with teeth taped together using autoclave tape can also be used to create larger wells.

N.B. We have run gels at 100V for 20 minutes and 70V for 60 minutes. The latter setting/time is better for running larger fragments such as whole plasmids as a high voltage can cause shearing of the DNA.

Isolating Plasmid from Cells (Miniprep)

Pellet overnight culture by centrifuging 13,000g for 10 minutes.
Pipette out supernatent and discard.
Add 250μl resuspension buffer R3 and mix by pipetting up and down.
Transfer to capped tubes.
Add 250μl lysis buffer L7 and mix by inverting tube 5 times.
Incubate for 5 minutes at room temperature.
Add 350μl precipitation buffer. Invert until mixture is homogenous.
Centrifuge 13,000g for 10 minutes. Place spin column into recovery tube.
Load supernatent into spin column and discard capped tube.
Centrifuge 13,000g for 1 minute. Discard supernatent.
Add 700μl wash buffer W9 (with ethanol).
Centrifuge 13,000g for 1 minute. Discard superantent.
Centrifuge 13,000g for 1 minute to remove all liquid. Discard any remaining supernatent and recovery tube.
Place spin column in new recovery tube.
Add 100μl TE buffer or MilliQ H2O.
Incubate for 1 minute at room temperature.
Centrifuge 13,000g for 2 minutes. The supernatent in the recovery tube should contain isolated plasmid.
Discard spin column and store plasmid at -20°C.

Restricting Plasmids (Double Restriction)

We have conducted restrictions in varying concentrations and total volumes; however they all follow the same basic procedure. Below are two of the different restrictions we carried out.

46μl MillQ H2O
10μl 10 x buffer
40μl plasmid sample
2μl enzyme 1
2μl enzyme 2

Total volume = 100μl

Concentrated

8μl MilliQ H2O
5μl 10 X buffer
10μl plasmid sample
1μl enzyme 1
1μl enzyme 2

Total volume = 30μl

Incubate solutions for 90 minutes in a 37°C water bath.
If DNA is to be run on a gel and a band cut out, this can be done without purifying the DNA. For all other downstream applications or for storage, solution must be purified to remove the enzymes.

Ligation

2.5μl of 10 x ligase buffer
6μl vector plasmid cut with relevant enzyme(s)
15μl of digested plasmid which contains the fragment to be inserted, digested with the same enzyme(s)as the vector plasmid
1.5μl of T4 ligase

= 25μl total volume

Control:

2.5μl of 10 x ligase buffer
6μl of vector plasmid cut with relevant enzyme(s)
15μl of MilliQ H2O
1.5μl of T4 ligase