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From 2013hs.igem.org

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CPEC
Wobble
EIPCR (enzymatic inverse PCR)
Normal ligation assembly (3A assembly)


Mini-preps
1) Resuspend pelleted bacterial cells in 250 µl (320 µL) Resuspension Buffer and transfer to a microcentrifuge tube.
2) Add 250 µl (320 µL) Lysis Buffer and mix by gentle inversion the tube 4–6 times to mix. Do not vortex, as this will result in shearing of genomic DNA
3) Add 350 µl (450 µL) Neutralization Buffer and invert the tube immediately but gently 4–6 times.
4) Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge.
5) Transfer the supernatant in a clean Eppendorf tube.
6) Add 0.6 volumes of isopropanol.
7) Centrifuge 10 minutes at maximum speed in a table top centrifuge
8) Wash with 70% ethanol.
9) Air dry and resuspend in 10 mM Tris pH = 7.4 (or ddH 2O)


Transformations
1.Thaw competent cells on ice.
2.Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube.
3.Add 50 µl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.
4.Place the mixture on ice for 30 minutes. Do not mix.
5.Heat shock at 42°C for 30 seconds*. Do not mix.
6.Add 950 µl of room temperature media* to the tube.
7.Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8.Warm selection plates to 37°C.
9.Spread 50–100 µl of the cells and ligation mixture onto the plates.
10.Incubate overnight at 37°C
* Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells’ manufacturer.


Ligations
1.Combine 50 ng of vector with a 3-fold molar excess of insert. Adjust volume to 10 μl with dH2O.
2.Add 10 μl of 2X Quick Ligation Buffer and mix.
3.Add 1μl of Quick T4 DNA Ligase and mix thoroughly.
4.Centrifuge briefly and incubate at room temperature (25°C) for 5 minutes.
5.Chill on ice, then transform or store at -20°C.
6.Do not heat inactivate. Heat inactivation dramatically reduces transformation efficiency.


Gel Digests
To cut DNA at specific sequences and often to leave sticky ends for ligating pieces together.

There are two types of Digests we do with restriction enzymes:
(1) screening/diagnostic of colonies
(2) digesting PCRs/plasmids for gel extractions & ligations.

(1) For diagnostic gels, we use between 50-200ng of DNA and digest for 1/2-1 hour before running on a gel. This is just to check if our DNA has the correct fragments
(2) For gel extractions, we use between 1500-3000ng of DNA and digest for 2-3 hours before running on a gel. The gel extraction procedure has low yield thus a lot is needed to start with.

Type (1) Diagnostic Gels
MIX: Typically 1.0-4.0 microliters DNA, 1.0 of each 10x buffer (check chart), 0.25-0.5 of each enzyme, fill up to 10.0 microliters total with water.
Leave at 37 incubator for 1/2-1 hour
Make gel with thin comb
By the time gel solidifies, probably ready to run gel
Typical Mix is
2.0 DNA
1.0 Buf 2
1.0 BSA
0.5 KpnI
5.5 H20
10.0 TOTAL

Type (2) Gel Extractions
Digest desired amount of DNA (will proba bly be around 30-60 microliters) for 2-3 hours at 37.
Make wide gel comb
Run gel till bands are well separated and cut out gel piece, trying to minimize amount of agarose
Typical Mix
46.0 DNA
6.0 Buf 2
6.0 BSA
1.0 KpnI
1.0 MluI
60.0 TOTAL
Note: After creating mix, vortex briefly, then spin down briefly to ensure consistency.
Note: Addition of BSA does not affect digests so if 1 enzyme requires it, just add it.




Normal PCRs
Gel-extractions
PCR-purifications
Running electrophoresis gels
Comp cells
Plating
Overnight cultures (liquid cultures)


Denote when the UCSD services had to be used – sequencing, designing primers, data managing techniques, ordering primers, thermocycler