Plasmid mini preparation kit | with 10ml DNA booster | 50 rxs
- Product Code: K-PMP5010
- Availability: In Stock
Tags: Plasmid preparation kit
Plasmid mini preparation kit | with 10ml DNA booster
Qty 50rxs (50 X column A*) + 5rxs (5 X column B**)
*/** Max DNA binding capacity for single columns: A: 50μg/rx | B: 80μg/rx
DNA recycling rate: A: 29μg/50μg (58%) | B: 38μg/50μg (76%)
Product uniqueness
- Media Enhancer (DNA Booster) for high-yield harvesting plasmids
- Equipped with a purification column with high DNA binding capacity and a high DNA recovery rate
Contains
- DNA booster (Ver1.2 | 10ml each for Low/High copy number plasmid) | Store at 4~6 ℃
- Buffer 1 (P1 suspension buffer/w RNase I) 15 ml | Store at 4~6℃ after adding RNase I
- Buffer 2 (P2 Denaturing fluid) 15 ml
- Buffer 3 (P3/N3 neutralisation buffer) 20 ml
- Washing Buffer A TA/IPA 22.5 ml
- Washing buffer B TA/EtOH 100 ml | Add 75ml pure ethanol before the first time use
- Elution Fluid (TE PH~8) 10 ml
- DNA Binding columns A 50 pieces
- DNA Binding columns B 5 pieces
- 2ml Centrifugal shell 50 pieces
- RNase I stock 500µL | Store at -20℃
Instruction
! Must do before the first time use
- Add 75ml pure ethanol into the Washing buffer B Bottle and shake well
- Add 10µL RNase I stock into the Buffer 1 Bottle and shake well, Store at 4~6℃ after adding RNase I
- Add 10µL RNase I stock into the Buffer 1 Bottle every 5~7 months, as RNase's activity is not stable in the P1 buffer.
Prepare in advance
- Water bath or metal bath the Elution Fluid at 50℃ (Optional)
- Prepare several clean DNase-free 1.5/2 ml centrifuge tubes (not included within the kit) for the final plasmid elution
- Prepare several 5 ml centrifuge tubes if the 15 ml Falcon tubes cannot be directly centrifuged in your machine.
Step 0 (Optional)
Add 5 ml DNA Booster to 245~250 ml autoclaved LB media, Aliquot 5 ml media each into 15 ml Falcon tubes, shake and incubate the bacteria at 37℃ overnight (8~14H, should not exceed 16 hours to avoid apoptotic lysis).
*The regular cultivation time can also be reduced by using DNA Booster.
Step 1.
Add 5 ml of cultured media into a 5 ml centrifuge tube, or directly centrifuge the Falcon tubes with media, centrifuge at 12,000 rpm for 30 s (or 4,000 rpm for 5min), and discard the supernatant.
Step 2
Add 250µL of Buffer 1 to the tube, then vortex until no visible bacterial particles are in suspension.
Step 3
Add 250μL Buffer 2 - Gently invert the tube 4~6 times until the liquid becomes transparent (this step should not take longer than 5 min).
Step 4
Add 350μL Buffer 3 - Gently invert the tube 6~10 times until no more yellow participants form (this step should not involve vigorous shaking).
Step 5
Centrifuge the 5ml centrifuge tube at 14,000 rpm / 3~5 min, and gently transfer the supernatant into the DNA binding column, and centrifuge the columns with a centrifuge shell at 12,000 rpm / 30s; or use a vacuum device to aspirate the liquid.
Step 6.1（Optional）
Discard the waste liquid within the centrifugal shell, insert the column back, then add 450 μL Wash Buffer A to the column to wash away unwanted protein contaminants (enzymes). To best clean the DNA, it is suggested to let the wash buffer soak the filter for about 3~5 min to dissolve the contaminants, then centrifuge the columns with centrifuge shell at 12,000rpm / 30s; or use a vacuum device to aspirate the liquid from the column.
*Must be washed as step 6.2 if step 6.1 has already been carried out, as IPA is difficult to dry.
Step 6.2
Discard the waste liquid within the centrifugal shell, insert back the column, and add 750~800μL Wash Buffer B into the column, to wash away unwanted salts and other contaminants. For the best purification of the DNA, it is recommended to let the Wash Buffer soak in the filter for about 3~5 min to maximally dissolve the salts. Then centrifuge the columns with a centrifugal shell at 12,000rpm / 30s; or use a vacuum device to to aspirate the liquid from the column.
*If using a vacuum device, the best time to stop is just after all visible liquid has been removed from the column. Over-vacuum will dry out the filter, which is detrimental to subsequent elution efficiency.
Step 6.3
*Repeat step 6.2 if the DNA is to be used for a salt-sensitive reaction such as PCR / RE digestion.
Step 7
Discard the waste liquid in the shell, put back the column, and centrifuge at 14,000 rpm for 1 min (or 12,000 rpm for 2.5 min) to remove all EtOH and prepare for DNA elution.
Step 8
Place the column in a clean DNase-free 2 ml centrifuge tube. Inject the appropriate amount of Elution Fluid into the centre of the filter to thoroughly wet all parts of the filter and dissolve the DNA (typically 50~100μL for thin filter columns, 100~150μL for thick filter columns). Allow standing at room temperature for 1 to 3 minutes, then centrifuge the columns together with the centrifuge tube at 12,000 rpm / 30 s and discard the column.
*We suggest eluting once by using 50~100μL Elution Liquid for Column A, which we have provided for fast plasmid preparation.
Elute twice by first using 100 µL and second using 50 µL Elution Fluid for Column B we provide, which aims to harvest more plasmid than the regular mini-preparation. This is because the thick filter may leave 20~25% DNA after the first elution compared to the thin filter (in column A) which leaves just under 10% after the first elution.
*Preheat the elution fluid to 50℃ before elution. This will give the best results.
*MQ water (PH~8, adjusted with Tris base) is also good for DNA elution.
*Column A can be reused (only for the same DNA in case of contamination) after 3~4 times 750µL MQ water (PH~8) elute (wash), column B suggested vacuum wash with 10ml warm MQ water (PH~8) before reuse.
