Plasmid construction

Table of Methods 6 December 2002 update 16 February 2018

PCR | Blunting | Ligation | Transformation

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 of 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!


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.

Isolation of DNA from gel bands:

You can cut out gel band using a 22mm coverslip (less dangerous and cheaper than a scalpel)
then use Qiagen gel band extraction kit: instructions

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.

Removing a restriction site with a 3' overhang: T4 polymerase "chew back" :

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)

Cloning from RT-PCR (Shana Fawcett)

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!).

Growing up colonies:
Use a yellow pipette tip and spear a colony.
Only take colonies that are well seperated from others on the plate.
Eject the tip into a sterile / capped glass tube
Add 5ml LB broth and 100mL of ampicillin* (100mg/ml stock)
Incubate in tube roller (third floor warm room) overnight.
* Note: if you yeild of plasmid DNA is poor, the most likely problem is that your ampicillin has "gone off". Make new ampicillin stock and stay aware of this possibility.
AMPICILLIN STOCK: 100mg/ml in distilled water

Wizard (Promega) minipreps:
1. Pellet 1.5ml of bacteria from an overnighter (3 min. spin in minifuge).
2. Discard supernatants / resuspend in 200mL resuspension buffer.
3. add 200mL cell lysis buffer - invert until clear.
4. add 200mL neutralization 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
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

Table of Methods