KIT 9040 50 PrimeWay Plasmid DNA Extraction Kit Manual

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com Molecular Biology Kits

PrimeWay Plasmid DNA Extraction Kit
Product No: KIT-9040-50
The PrimeWay Plasmid DNA Extraction Kit is a rapid and reliable kit used to purify
high quality plasmid DNA. It uses the alkaline lysis method to purify plasmid DNA
from bacteria. This kit also comes with RNase A to remove RNA during extraction. It
uses a silica-based spin column method and is suitable for extracting plasmid DNA
up to 15 kb within 25 minutes. The purified plasmid is suitable for downstream
application such as DNA sequencing, PCR, in vitro transcription, restriction mapping,
cloning and DNA labeling applications.
For Research Use Only. Not for use in Diagnostic Procedures.
Kit Contents
No Product KIT-9040-50 Storage
1 pD1 Buffer 15 mL
2 pD2 Buffer 15 mL
3 pD3 Buffer 20 mL
4 Wash Buffer A1 16 mL
Room temperature
5 Wash Buffer A2 8 mL
(21 oC – 25 oC)
6 Elution Buffer 5.5 mL
7 RNase A 1.5 mg
8 PrimeWay Plasmid Column 50 pcs
9 Collection Tube 50 pcs
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Product Specification
KIT-9040-50
Binding capacity 60 µg
Yield Up to 40 µg
Sample 1 – 5 mL bacterial culture
Plasmid size Up to 15 kb
Elution 50 - 100 µL
Duration < 25 minutes
Materials Supplied by Users

 Ethanol (96 – 100%)
 Centrifuge, at speed of 18,000 × g
 Vortex mixer
 1.5 mL microcentrifuge tubes
Precautions for Users

 Some buffer of this kit contains irritants. Handle with care and avoid contact
with skin. In case of contact, wash skin with plenty of water and seek medical
attention.
 Always wear a lab coat, disposable gloves and surgical mask.

Protocol – Plasmid DNA Extraction
I. Add 500 µL of pD1 Buffer to RNase A and vortex to dissolve. Short spin
to bring down the volume and transfer the RNase A solution to pD1
Buffer. Mix well and store pD1 Buffer at 4 °C
Preparation
II. Add 6 mL ethanol (96 – 100%) into Wash Buffer A1. Mix well before use.
III. Add 32 mL ethanol (96 – 100%) into Wash Buffer A2. Mix well before
use.
IV. Make sure no precipitation observed in pD2 Buffer. Dissolve the

precipitate by warming the pD2 Buffer in water bath at 37 °C.
1. Pellet the cells by centrifuging it at 11,000 x g for 1 minute.

Sample
2. Discard the supernatant.
3. Resuspend the cell pellet with 250 µL pD1 Buffer by vortexing or

pipetting up and down until all cell pellet dissolved.
Note: Make sure that RNase A has been added into the pD1 Buffer.
4. Transfer the cell suspension to a new 1.5 mL microcentrifuge tube.
5. Add 250 µL pD2 Buffer, mix by gently inverting the tube for 5 – 10 times.
6. Incubate the lysate mixture at room temperature for 2 – 5 minutes until

Lysis
the lysate become clear.

Note: Do not vortex to avoid shearing of genomic DNA
Do not incubate more than 5 minutes.
7. Add 350 µL pD3 Buffer to neutralize the lysate. Mix immediately by

inverting the tube 5 – 10 times.
8. Centrifuge at maximum speed (~ 18,000 × g) for 10 minutes to pellet the

cell debris.

9. Place the PrimeWay Plasmid Column into a Collection tube
10. Transfer the clear supernatant into the PrimeWay Plasmid Column.
Binding
Note: Do not transfer any white pellet into the column
11. Centrifuge at 11,000 × g for 30 seconds. Discard the flow-through and

place the column back to the Collection tube.
12. Add 400 µL Wash Buffer A1 into the PrimeWay Plasmid Column.
Important!! Make sure ethanol is added to the buffer prior to first use.
13. Centrifuge at 11,000 × g for 30 seconds and discard the flow-through.

Place the column back to the Collection tube.
Washing
14. Add 700 µL Wash Buffer A2 into the PrimeWay Plasmid Column.
Important!! Make sure ethanol is added to the buffer prior to first use.
15. Centrifuge at 11,000 × g for 30 seconds and discard the flow-through.

Place the column back to the Collection tube.
16. Centrifuge the column at maximum speed (~ 18,000 × g) for 3 minutes

to remove Wash Buffer residual.
Drying
Note: Make sure the residual liquid is completely removed as it can

inhibit downstream application.
17. Place the PrimeWay Plasmid Column into a new 1.5 mL microcentrifuge
tube.
18. Add 50 – 100 µL Elution Buffer to the center of the PrimeWay Plasmid
Elution
Column membrane.
19. Incubate at room temperature for 1 minute and centrifuge at maximum

speed (~ 18,000 × g ) for 1 minute to elute the plasmid DNA.
20. Store the extracted plasmid DNA at -20 °C

Troubleshooting Guidelines
Problems Possible Reason Recommended Action

Low plasmid Poor lysis of bacteria Ensure the cell pellet is completely resuspended
yield cells before lysis. No cell clumps should be visible.
Insufficient of bacterial Grow the bacteria cells longer at 37°C under
cells shaking but not more than 16 hours
Bacteria culture is too Do not overgrown the bacterial cells for more than
old 16 hours at 37 °C under shaking.
DNA Elution Ensure that the Elution Buffer is added to the
center of column membrane and completely
absorbed by the column matrix
Wash Buffer was not Ensure ethanol has been added to Wash Buffer A1
prepared accordingly and Wash Buffer A2 accordingly before use.
Presence of Absence/ Insufficient Ensure that all the dissolved RNase A is added into
RNA volume of RNase A pD1 Buffer.
contamination
Problem in the Ethanol contamination Increase centrifugation time with additional 3
downstream minutes to ensure the Wash Buffer is completely
processes removed.
Genomic DNA Vigorous lysis/ long lysis • During lysis step, gently invert the solution to
contamination incubation time mix, do not vortex.
• Do not incubate the lysis mixture for more
than 5 minutes
Please contact us at https://base-asia.com/contact/ for more information.

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www.base-asia.

com Molecular Biology Kits

For Research Use Only. Not for use in Diagnostic Procedures.

Ver. 1.0 Page 1 of 5 www.base-asia.com

Materials Supplied by Users

Precautions for Users

Ver. 1.0 Page 2 of 5 www.base-asia.com

IV. Make sure no precipitation observed in pD2 Buffer. Dissolve the

1. Pellet the cells by centrifuging it at 11,000 x g for 1 minute.

2. Discard the supernatant.

3. Resuspend the cell pellet with 250 µL pD1 Buffer by vortexing or

4. Transfer the cell suspension to a new 1.5 mL microcentrifuge tube.

6. Incubate the lysate mixture at room temperature for 2 – 5 minutes until

the lysate become clear.

7. Add 350 µL pD3 Buffer to neutralize the lysate. Mix immediately by

8. Centrifuge at maximum speed (~ 18,000 × g) for 10 minutes to pellet the

Ver. 1.0 Page 3 of 5 www.base-asia.com

Note: Do not transfer any white pellet into the column

11. Centrifuge at 11,000 × g for 30 seconds. Discard the flow-through and

13. Centrifuge at 11,000 × g for 30 seconds and discard the flow-through.

15. Centrifuge at 11,000 × g for 30 seconds and discard the flow-through.

16. Centrifuge the column at maximum speed (~ 18,000 × g) for 3 minutes

Note: Make sure the residual liquid is completely removed as it can

19. Incubate at room temperature for 1 minute and centrifuge at maximum

20. Store the extracted plasmid DNA at -20 °C

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Problems Possible Reason Recommended Action

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