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GB-Clean™ Plasmid Miniprep Kit

GB-Clean™ Plasmid Miniprep Kit is designed for rapid and cost-effective small-scale preparation of high quality plasmid DNA from recombinant E.coli cultures. The kit utilizes a silica-based membrane spin column. The kit can be successfully used for efficient purification of any size plasmids and cosmids. The input culture volume and yield depend on the plasmid copy number and growth conditions. 

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CAD$50.00

  • 50 preps
  • 100 preps
  • 200 preps

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GB-Clean™ Plasmid Miniprep Kit is designed for rapid and cost-effective small-scale preparation of high quality plasmid DNA from recombinant E.coli cultures. The kit utilizes an exclusive silica-based membrane technology in the form of a convenient spin column. Each spin column can recover up to 40 μg of plasmid DNA. The kit can be successfully used for efficient purification of any size plasmids and cosmids. The actual plasmid yield and optimal culture volume depend on the plasmid copy number and medium used for cultivation.

Principle
Pelleted bacterial cells are resuspended and subjected to SDS/alkaline lysis (1) to liberate the plasmid DNA. The resulting lysate is neutralized to create appropriate conditions for binding of plasmid DNA on the silica membrane in the spin column (2). Cell debris and SDS precipitate are pelleted by centrifugation, and the supernatant containing the plasmid DNA is loaded onto the spin column membrane. The adsorbed DNA is washed to remove contaminants, and is then eluted with a small volume of the Elution Buffer (10 mM Tris-HCl, pH 8.5).The purified plasmid DNA is ready for immediate use in all molecular biology procedures such as conventional digestion with restriction enzymes, PCR, transformation and automated sequencing.
References
1. Birnboim H.C., and Doly, J. (1979) A rapid alkaline lysis procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513 -1522.
2. Vogelstein, B. and Gillespie, D. (1979) Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76, 615-619.

Available in 50, 100 and 200 prep sizes

Kit content (for 50 prep kit size)

Component

Volume or Size

RNase A

150 µl

Resuspension Solution

15 ml

Lysis Solution

15 ml

Neutralization Solution

20 ml

Wash Buffer (PB)

30 ml

Wash Buffer (W)

30 ml

Elution Buffer (10 mM Tris-HCL, pH 8.5)

5 ml

Spin Columns

50 each

Caution: * Wear gloves when handling Neutralization Solution.

Warning: *Do not add bleach to sample preparation waste.
Storage and stability

• Store RNase A at -20.

• Store High Purity Plasmid Miniprep Kit except RNase A for up to 12 months at room temperature (15-25) or at for storage periods longer than 12 months 4 .

• If precipitate forms in the buffers during storage, it should be re-dissolved by incubating the buffers at 37 for 3 min before use.

Important notes
Add the provided RNase A solution to the Resuspension Solution and mix. After addition of RNase A, the Resuspension Solution can be used for 6 months when stored at 4.
Prior to the initial use of the kit, add ethanol (100%) (not included in the kit) to the Wash Buffer (W)

50 prep kit

100 prep kit

200 prep kit

Washu Buffer (W)

30 ml

30 ml x2

40 ml x2

Ethanol

45 ml

45 ml x2

60 ml x2

Total Volume

75 ml

75 ml x2

100 ml x2


After the ethanol has been added, make sure to mark the check box on the bottle to indicate the completed step.
Visually inspect the Lysis Solution and the Neutralization Solution for salt precipitation before each use. Re-dissolve any precipitate by warming the solution at 37, then cool back down to 25 before use. Do not shake the Lysis Solution too vigorously.
Both the Lysis Solution and the Neutralization Solution contain irritants. Wear gloves when handling these solutions.
All purification steps should be carried out at room temperature.
All centrifugations should be carried out in a table-top microcentrifuge at >12000x g (10,000-14,000 rpm, depending on the rotor type).

Growth of Bacterial Cultures
1. Pick a single colony from a freshly streaked selective medium plate to inoculate 1-5 ml of LB medium supplemented with the appropriate selection antibiotic. Incubate for 12-16 hours at 37°C while shaking at 200-250 rpm. Use a tube or flask with a volume of at least 4 times the culture volume to provide sufficient aeration.
2. Harvest the 1-10 ml bacterial culture by centrifugation at 8000 rpm (6800x g) in a microcentrifuge for 2 min at room temperature. Decant the supernatant and remove all remaining medium.
Warning: Do not overload the column:
For high-copy-number plasmids, do not process more than 5 ml of bacterial culture (see Table 1). If more than 5 ml of such a culture are processed, the spin column capacity (40 µg of dsDNA) will be exceeded and no increase in plasmid yield will be obtained.
For low-copy-number plasmids (see Table 1), it may be necessary to process larger volumes of bacterial culture (up to 10 ml) to recover a sufficient quantity of DNA.

Table 1 Copy numbers of various vectors

High-copy number (300-700 copies/cell)

Low-copy number (10-50 copies/cell)

Very low-copy number (up to 5 copies/cell)

pUC vectors

pBluescript vectors

pGEM vectors

pTZ vectors

pJET vectors

pBR 322 and derivatives

pACYC and derivatives

pSC101 and derivatives

Procedure

1. Pellet 15 ml of an overnight culture (12 x 109E.coli in LB medium with a selective antibiotic) in an appropriate tube. Thoroughly remove all medium from the cell pellet.

2. Resuspend the pelleted cells in 250 μl of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
Note. Ensure RNase A has been added to the Resuspension Solution.

3. Add 250 μl of the Lysis Solution and mix content thoroughly by inverting the tube 4-6 times until the solution becomes viscous and clear.
Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 minutes to avoid denaturation of supercoiled plasmid DNA.

4. Add 350 μl of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate is cloudy and viscous.

5. Centrifuge for 10 min at 14,000 × g (12,000 rpm) to pellet cell debris and chromosomal DNA.

6. Transfer the supernatant to the supplied spin column (mounted on a collection tube) by decanting. Avoid disturbing or transferring the white precipitate.

7. Centrifuge for 1 min. Discard the flow-through by emptying the liquid in the collection tube and place the column back into the same collection tube.
Note. Do not add bleach to the flow-through.

8. Add 500 μl of the Wash Solution (PE) to the spin column. Centrifuge for 30-60 seconds at 14,000× g (12,000rpm) and discard the flow-through from collection tubes. Place the column back into the same collection tube.

9. Add 500 μl of the Wash Solution (W) to the spin column. Centrifuge for 30-60 seconds at 14,000× g (12,000 rpm) and discard the flow-through. Place the column back into the same collection tube.

10. Repeat the wash procedure (step 9) using 500 μl of the Wash Solution (W).

11. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.

12. Transfer the spin column into a fresh 1.5 ml microcentrifuge tube (not included in the kit). Add 50 μl of the Elution Buffer to the center of spin column membrane to elute the plasmid DNA. Take care not to touch the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min at 14,000× g (12,000rpm).
Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70 before applying the EB to silica membrane.

13. Discard the column and store the purified plasmid DNA at -20.

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