Key points to observe:
a. Use a endA1- E. coli strain for plasmid propagation and isolation whenever possible. The instability of plasmids isolated from endA l + bacterial strains has been reported (Schoenfield et al., 1995).
b. Do not vortex, shake or incubate for more than 5 min in step 3. This may cause shearing of genomic DNA and/or linerization (or unravelling) of the supercoiled plasmid. A Iysis time of less than 5 min is important to cause maximum release of plasmid while minimising plasmid denaturation. The lysate should be clear and viscous.
c. Use of cold room or less than 7 min centrifugation may give rise to a dirty supernatant in step 4. If for whatever reason the centrifugation has to be performed at low temperature, the mixture should be transferred to room temperature as quickly as possible after centrifugation.
d. In earlier protocols and in protocols of commercial miniprep plasmid purification kits, less than 1 min centrifugation is recommended to remove ethanol from either the binding resin or a diatomaceous earth column, but we found that under these conditions some ethanol still remained in the diatomaceous earth. Therefore, centrifugation should be at least 3 min in step 7. If necessary, repeat the centrifugation twice. DNA will not be lost.
e. Use only half of the first volume during step 9. If 100 'micro'l is used for the first elution, then we recommend less than 50 'micro'l for the second elution. If the diatomaceous earth is found in the bottom of the tube following centrifugation, transfer the supernatant carefully into a new eppendorf tube.
Troubleshooting and Hints
(i) Very low yields of plasmid - this is usually attributed to a loosely fitting filter in the column. Check whether the filter in the column is fitted lightly. Check the plasmid copy number. Was antibiotic added or not?
(ii) Low purity of plasmid with an OD 260/280, greater or less than 1.8 - 2.0. This usually arises from white gelatinous matter remaining above the diatomaceous earth when preparing the solution. Check the diatomaceous earth solution. Check whether endA 1-/+ cells were used.
(iii) Vacuum is best applied from a steady source such as 'house vacuum'. Syringes tend to stick and give bursts of vacuum.
(iv) If the supernatant in step 5 contains cell debris in suspension because of careless transfer, the column will clog. In this case do not discard sample, but scratch column surface slightly with pipette tip to unclog column.
(v) Cheap ICN Practical Grade guanidine hydrochloride is quite suitable, as long as undissolved solids are removed by filtration once the theoretically 6M solution has been made up.
(vi) Home made filter columns can be made using microcentrifuge tubes as described by Hansen et al. (1995), but we recommend piercing the bottom of the tube with a needle, from the inside, rather than snipping the bottom off.
(vii) In principle it should be possible to scale this up to a macro-prep. We have only worked with 20 ml cultures per prep.
References:
1. Birnboim, H. C. 1983. A rapid alkaline extraction method for the isolation of plasmid DNA.Methods Enzymol. 100, 243 - 255.
2. Hansen, Nils Jakob V., P. Kristensen, J. Lykke, K. K. Mortensen and B. F. C. Clark. 1995. A fast, economical and efficient method for DNA purification by use of a homemade beads column.Biochemistry and Molecular Biology Intemational 35 (3), 461 - 465.
3. Sambrook J. et al., 1989. Molecular Cloning; A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
4. Schoenfeld, T., J. Mendez, D. R. Storts, E. Portman, B. Patterson, J. Frederiksen and C. Smith. 1995. Effects of bacterial strains carrying the endAI genotype on DNA quality isolated with Wizard Plasmid Purification System. Promega Notes, 53, 12 - 22.
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Triton-Prep Method for bacterial DNA Purification
Grow 5 ( large scale-15ml culture). Harvest in single eppendorf tube (or 15 ml disposable tube).
Resuspend pellet with 300ul STET buffer (900ul). After resuspending add 30ul RNase/lysozyme mixture (100ul).
Boil for one minute 15 seconds (one minute 45 seconds).
Spin in microfuge for at least 15 minutes.
Take supernatant and phenol extract with 150ul (500ul) STET- saturated phenol.
Spin and take supernatant. Add 1/10 volume 4M lithium chloride (autoclaved). Let sit on ice for 5-10 minutes.
Spin and take supernatant. Add equal volume isopropanol. RT for 5 minutes.
Spin. No pellet will be visible. Don't panic, DNA is stuck to side all the way up tube.
Important: Wash with 80% ethanol (95% will cause the residual Triton to precipitate)
Resuspend pellet in 50-200ul.
Lysozyme/ RNase mixture
10mg/ml lysozyme
1mg/ml RNase (use cheap grade (BMB) rather than RNase A , which is too expensive)
50mM Tris-HCl pH8.0
Store at -20oC in small aliquots. Do not refreeze after thawing.
STET
8% sucrose
5% Triton X-100
50mM Tris-HCl (pH8.0)
50mM EDTA pH 8.0
Filter sterilize. Store at 4oC
[NextPage] Cultures should be grown in LB for best results. To avoid overloading the Qiagen columns, the correct number of cells/volume of culture should be determined. Qiagen recommends not more than 4x10E9 cells for a mini-column, 2x10E10 for the midi, and 8x10E10 for the maxi. For this is ~0.4-0.8 ml, 2.0-4.0 ml, and 10.0 to 20.0 ml for overnight cultures in LB. 1. Prepare buffers according to recipe at end. Equilibrate all to room temp. before use. 2. Dissolve Rnase A in buffer B1 to concentration of 200 ug/ml. Stock solutions of lysozyme and proteinase K can be made in dH2O to concentrations of 100 mg/ml and 20 mg/ml respectively. 3. Pellet cells by centrifugation at 3000-5000x g for 5-10 min. Remove supernatant. 4. Resuspend the bacterial pellet in 1/3.5/11 ml (mini/midi/maxi columns) of Buffer B1 by vortexing at top speed. 5. Add 20/80/300 ul of lysozyme stock solution and 45/100/500 ul of proteinase K. Incubate at 37 C for at least 30 min. 6. Add 0.35/1.2/4 ml of Buffer B2 and mix by inversion several times. Incubate at 50 C for 30 min. Mix well; this step is important for efficient deproteinization. It is also important that the lysate becomes clear at this stage. 7. Equilibrate Qiagen genomic tip with 2x1/4/10 ml Buffer QBT, and allow the tip to empty by gravity flow. 8. Vortex the lysate for 5-10 sec and apply onto the equilibrated column. Again, allow it to pass through column by gravity flow. Flow can be assisted with gentle positive pressure (a plunger from a 10 ml syringe fits the midi columns) but it is also OK to dilute the lysate with an equal volume of Buffer QBT prior to loading. The latter is preferable by virtue of personal experience. 9. Wash the column with 3x1 ml/2x7.5ml/2x15 ml of Buffer QC. Allow buffer to pass through by gravity flow. Two washes should be enough. 10. Elute the genomic DNA with 2x1 ml/5 ml/15 ml of Buffer QF. Precipitate with 0.7 volumes (1.4/3.5/10.5 ml) isopropanol, equilibrated to room temperature. The DNA should be spoolable; if not, pellet the precipitate by centrifugation at 5000+ x g. Wash the precipitate with 70% EtOH and dry. Resuspend in the appropriate solvent (TE, water).Bacterial Genomic DNA Purification via Qiagen columns
Amount/prep Mini Midi MaxiB1 1 ml 3.5 ml 11 ml
B2 0.35 ml 1.2 ml 4 ml
QBT 2 ml 4 ml 10 ml
QC 3 ml 15 ml 30 ml
QF 2 ml 5 ml 15 ml
RNaseA 0.2 mg 0.7 mg 2.2 mg
lysozyme 2 mg 8 mg 30 mg
Qiagen protease* 0.9 mg 2 mg 10 mg
*or proteinase K
Solutions:
Buffer Composition (Storage)
B1 50 mM EDTA, 50 mM Tris/HCl, 0.5% Tween 20, 0.5%
Triton X-100 (room [4 C after addition of RNase])
B2 3 M GuHCl, 20% Tween 20 (room temp)
QBT 750 mM NaCl, 50 mM MOPS, 15 % ethanol, 0.15% triton
X-100, pH 7.0 (room temp.)
QC 1.0 M NaCl, 50 mM MOPS, 15% ethanol, pH 7.0 (room temp.)
QF 1.25 M NaCl, 50 mM Tris/HCl, 15 % ethanol, pH 8.5 (room temp.)
Chromosomal DNA Extraction from Gram-positive Bacteria
This procedure was originally developed for Listeria monocytogenes but has worked well with other Gram+ bacteria we've tried.
Pellet cells from 10 ml overnight cultures in BHI or LB and wash in 5 ml of 0.1X SSC.
Resuspend in 1 ml 10 mM Tris-HCl (pH 8.0) containing 20 % sucrose (v/v), add lysozyme to 2.5 mg/ml, and incubate at 37 C for 45 min.
Add 9 ml lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 500 mg pronase B, 1 % SDS), and incubate additional 30 min at 37 C.
Phenol and chloroform extract lysed cells, and ethanol precipitate the DNA with 0.1 vol. 3 M sodium acetate, pH 4.8 and 2 vol. 95% ethanol.
Spool out DNA with a glass rod, wash once with 80% ethanol before drying.
Some bacterial species may require a longer incubation in lysozyme. For Renibacterium salmoninarum (a G+ salmon pathogen we work with), lysozyme incubations overnight at 37 C worked very well with high yields of DNA
ref: Flamm, R. K., Hinrichs, D. J., and Thomashow, M. F. 1984. Introduction of pAMb1 into Listeria monocytogenes by conjugation and homology between native L. monocytogenes plasmids. Infect. Immun. 44:157-161.
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