Checking your plasmids & Restriction Digests

Simple diagnostic digests to determine if you are dealing with the correct plasmid.

Taking DNA from a standard miniprep,
you would normally analzye 2µL of uncut DNA and 5µL a restriction digest.

Standard digest

• 5 µL of plasmid DNA
• 3 µL of appropriate buffer. Each enzyme has its own optimal buffer -- check chart to determine which is best for the enzyme(s) you are using.
• 1 µL of each enzyme (never more than 10% enzyme!)
• water to 30 µL

Always check that you know the temperature at which the enzymes cuts!

Standard conditions, 1 hour @ 37°C.

ALWAYS KEEP ENZYMES IN THE BIG PIG, return to -20°C as soon as you are done!

CHANGE PIPETTE TIPS TO AVOID CROSS-CONTAMINATING ENZYMES

Partial digests:

In some cases you may need to do a partial digest. This can be tricky.
Always carry out a complete digest to determine the final pattern.
A good starting point is to use the enzyme (1µL) diluted 1:10 in its appropriate buffer + acetylated BSA) and reduce the time and temperature of the incubation.

I suggest 5-10 min. at r.t.

DNA gel analysis:

For a large gel: use 0.6-0.8 g agarose melted into 100ml TAE buffer
add 10µL 1mg/ml Ethidium bromide solution

Dissolve in microwave - 85-90 seconds

COOL solution to below 60°C before pouring gel

Run gel at betwee 70-100V.

NOTE: Gels can be stored in the cold room for a day or two, so if you pour a large gel, it can be used to analyze a number of samples.

Removing a restriction site with 5' overhang: Klenow fill-inreaction:

Following restriction reaction add

5uL of 2.5 mM dNTPs & 5 µL of Klenow enzyme.
Incubate at room temp for 20 minutes and then for 5 minutes at 37°C.
ligate and transform.

Polymerase chain reaction:

This is my standard set up for simple amplification from a plasmid

1 to 5 µL of plasmid (miniprep) DNA
30 µL nucleotides
20 µL polymerase buffer
2 µL of each primer (1 µg/µL)
1 µL MgSO4 stock (100mM)
2 µL Vent

water to 200 µL - run as 50 µL samples (4 tubes)

Ligation reaction:

Take DNA (plasmid + insert or whatever) isolated from gel bands

resuspend in 25 µL water (27 uL to elute Quiagen column, 2 µL will be lost)

add 3 µL T4 ligase buffer and 2µL T4 DNA ligase

incubate > 2h at 16°C and then transform into competant bacteria.

Standard transformation:
1. add 1mL of miniprep DNA or 15mL of a ligation reaction to a vial of competant cells
2. let sit on ice for 30-180 minutes.
3. transfer to a plastic-capped tube / heat shock at 42°C for 1 min or 37C for 5 min.
4. add 0.8 ml LB media - let grow for more than 20 minutues at 37°C
5. plate out onto LB + AMP plates

typically for a simple transformation of cells with intact plasmid, I would make
two plates, using 50uL and the other with 200uL of cells.
for a ligation reaction, I would plate the entire transformation onto two plates.
Always flame the glass spreader and allow it to cool before using it to spread cells out on a plate. If it is too hot, it will kill the cells.
If you do not flame it, you will cross contaminate cultures (a bad thing to do!).