CsCl gradient centrifugation for
Maxipreps.Long protocol Plasmid preps DNA purification
Contributor:: Zappe, H
Note: Refer to Sambrook et al. for safety regarding the use of Ethidium
Bromide. 
Plasmid prep: Ish-Horowicz
adaptionShort mini-prep protocol Long protocol Related Contents
Description:
Mini- midi- and Maxi- plasmid preparation
(Based on Ish-Horowicz and Burke. N.A.R. 9:2989-2998 (1981), Birnboim and Doly N.A.R. 7:1513-1523 (19??), BRL Labs with modifications for CsCl spin. The method is similar to that described starting on page 1.38, Sambrook et al., with modifications). Refer to Chapter 1, page 1.21 of Sambrook et al. for further reading.
Plasmid prep: Ish-Horowicz
adaptionShort protocol Related Contents
Contributor:: Zappe, H
Reference: Based on Ish-Horowicz and Burke. N.A.R. 9:2989-2998 (1981), Birnboim and Doly N.A.R. 7:1513-1523 (19??), BRL Labs with modifications for CsCl spin. The method is similar to that described starting on page 1.38, Sambrook et al., with modifications). Refer to Chapter 1, page 1.21 of Sambrook et al. for further reading.
1. From a single colony on a Luria agar plate, inoculate a 5 ml Luria broth (LB) starter culture with antibiotic selection and incubate to late log (OD600 = 0.6 - 0.8, 6 - 8 h). Dilute the starter culture 1/100 to the main culture (see note) and shake O/N (16 h). Ampicillin selection at 100 mg /ml.
Prep type: miniprep midiprep maxiprep
Culture vol. 1.5-4 ml 50 ml 400 ml
Use tube microfuge J21 - 50 ml 500 ml
Note: For maxipreps, usually 200 ml of Luria broth in a 1 liter flask will yield plenty of plasmid (e.g high copy number plasmids such as pUC or Bluescript) but for lower copy number plasmids like pBR322, larger culture volumes of a richer medium may be necessary and/or the plasmids can be amplified by the addition of 170 - 200 mg/ml of Chloramphenicol to the main culture when to OD600 reaches about 0.5. Continue to shake to culture for a further 12 - 16 h before harvesting (see chapter 1, Sambrook et al.)
2. Harvest the cells by centrifugation in appropriate rotor (sufficient speed and time to pellet the cells). Pour off the spent broth to the culture flask, invert the tubes on paper towel to remove the last of the broth and then resuspend completely in the indicated volume of Solution I:
miniprep midiprep maxiprep
Add 0.2 ml 4.0 ml 32 ml
3. Add solution 2, mix well by shaking and leave on ice for 5 min. (Culture should clear and go gloopy). This is a timed denaturation step - do not exceed 5 min.
Add 0.4 ml 8.0 ml 64 ml
4. Add pre-cooled solution 3, shake well and leave at least 5 min on ice:
Add 0.3 ml 6.0 ml 48 ml
Note: A white flocculate forms - like curds and whey - cellular protein and membrane precipitate.
5. Spin 10 min (12 K), recover indicated supernatant and transfer to fresh tubes of the same volume. Avoid contamination with pelleted material (Larger volume preps. can be filtered through cheesecloth):
Recover: 0.9 ml 18.0 ml 144 ml
6. Add 0.6 ml volumes of isopropanol, mix, leave for 10 min, then spin for 5 - 10 min (12 K for J-21, 8K for GSA).
Add 0.6 ml 10.0 ml 86 ml
7. Discard the supernatant, wash the pellet in 80 % ethanol, drain thoroughly (i.e. pour off the ethanol, spin the tube briefly and then remove the last drops with a pipette), and resuspend the pellet in TE:
Add 0.3 ml* 1.0 ml 7.5 ml
* If the miniprep is just going to be used for checking for an insert by restriction, reduce this volume to 50 ‘l, and use 1 ‘l in a digest.
The method for maxipreps can now be continued in one of a number of ways: CsCl gradient centrifugation, RNase treatment and phenol extraction (both given below), selective precipitation with polyethylene glycol (page 1.40, Sambrook et al.), or using NucleobondŸ column purification.
ice
isopropanol
ethanol
Mini-prep Notes - no phenol
extraction
Contributor:: Zappe, H
RNase treatment and phenol purification for mini,
midi, and maxi preps.Related Contents Long protocol Plasmid preps
Contributor:: Zappe, H
1. Continue with the volumes as in step 7 of the maxiprep protocol, except make maxiprep to 9 ml TE, and add the appropriate vol. of RNase solution (1 mg/ml heat treated RNase A; 4000 U/ml RNase T1) and incubate at 37 C for 15 min.
Add 20 ml 0.2 ml 0.5 ml
2. Add phenol/CHCl3/8-hydroxyquinoline mixture, mix, spin 12 K for 10 min, recover aqueous phase to clean tube.
Add 0.2 ml 0.2 ml 4.0 ml
3. Add 0.5 volumes of cold 7.5 M NH4-acetate, mix and leave on ice for 15 min. Spin 12 K for 10 min, transfer supernatant to fresh tube, discard pellet.
Add 0.15 ml 0.6 ml 4.5 ml
4. Add 0.6 volumes of isopropanol, leave on ice for 10 min, spin 12 K for 15 - 20 min.
Add 0.3 ml 1.1 ml 8.5 ml
5. Wash pellet with cold 80% ETOH, drain properly or spin briefly and remove last drops of ETOH.
6. Dissolve DNA in TE.
Add 50 ml 0.2 ml 0.5 ml
7. Store in aliquots at -20 C or better at -70 C
Mini-prep method - short
protocolRelated Contents Long protocol
Contributor:: Zappe, H
Plasmid prep: JAT prepDescription:
I received the method from Helen Stutz and I believe it to originate from Prof JA Thomson. There is no reference available for the method. It is a very quick way to test whether a E. Coli colony contains a plasmid with an insert. The DNA is electrophorezed without digestion and ccc (closed-circular covalent) DNA of comparitive size is run as a control.
Plasmid prep: JAT preps,
protocolContributor:: Stutz, H
Reference: ?