Polymerase chain reaction:  

here is a good on-line reference for PCR amplification

100 µL reaction (two 50µL tubes) - for subcloning - for PCR verification you need only 50 µL reaction.

10 µL 10XThermoPol buffer
1.6 µL mixed dNTPS (each at 25 mM)
1 µL each forward and backward primers (from 100 µL stocks)
1 µL plasmid
1 µLVent enzyme waterto 100 µL, 50 µL per tube

Run program Vent1
- adjust annealing time 5ºC belong lowest primer Tm
- extension time 1 min per 1000 base pair lenght.
- 30 cycles / hold overnight at 4ºC - take in the morning
- do not leave machine running more than 18hours.

You can
check PCR by running 2.5-5 µL on gel
cut the PCR using CutSmart buffer - cut 3 to 5 µL of PCR reaction (should be plenty) in 30 to 40 uL reaction

This is my old 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)

Checking your plasmids & restriction digests

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

Since this was originally written NEB has introduced CutSmart buffer - which often is the best choice for single and double digests (check the back of the NEB catalog)..  

Taking DNA from a standard miniprep,
you would normally analzye 2µL of uncut DNA and 4 to 5µL of a restriction digest.

Standard digest

• 3 to 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.

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.

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 1µL of miniprep DNA or 15µL 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!).

GeneJet  protocol for minipreps 

1. Pellet 1.5ml of bacteria from an overnighter (3 min. spin in minifuge).
2. Discard supernatants / and add another 1.5 ml of culture - spin down and discard supernatant
3. resuspend in 250 mL resuspension buffer (use Alberto's trick)
3. add 250 µLcell lysis buffer - invert until clear.

4. add 350 µL neutralization buffer - invert to mix and then spin in microfuge for 5 minutes.

5. take supernatant - transfer to a GeneJet column (tube) tube
6. centrifuge 1 min.  - discard flow through

7. Wash with 500 µL - spin and discard flow through
8. Wash again with 500 µL wash
9. spin on more time

10. take mini-column and place in microfuge tube add 50 µL of elution buffer and let sit for 2 minutes.
11. centrifuge to collect DNA (plasmid).

label your tube - plasmid all tubes in labelled plastic bag with plasmid name, date, your name and whether it has been confirmed by PCR or digest - plasmid can be stored frozen. . 

Wizard (Promega) minipreps:

1. Pellet 1.5ml of bacteria from an overnighter (3 min. spin in minifuge).
2. Discard supernatants / resuspend in 200 µLresuspension buffer.
3. add 200 µLcell lysis buffer - invert until clear.
4. add 200 µLneutralization buffer - invert and then spin in microfuge for 5 minutes.
5. take supernatant - transfer to a new tube then add 0.8ml of resin
- invert and let sit for 1 min.
6. attach mini-column to 3ml syringe / or manifold (syringes can be reused!)
and load with resin/cell supernatant.
7. push material through the mini-column
8. remove syringe, pull out plunger, replace syringe. add 2ml wash solution -
push through the mini-column.
10. take mini-column and place in microfuge tube - centrifuge for 20 sec.
11. take mini-column and place on fresh tube, add 50mL 65°C water, centrifuge again for 20 sec. -- your DNA is in the microfuge tube!

Resuspension buffer: 50mM Tris pH 7.5
10mM EDTA
100mg/ml RNAase A

Lysis buffer: 0.2M NaOH
1% SDS

Neutralization buffer: 1.32 M Kacetate pH 4.8

Ethanol wash buffer: 0.2M NaCl
20mM Tris pH 7.5
5mM EDTA ADD 70ml 100% ethanol / 50ml wash buffe