Index

I. Plasmid Purification
II. PCR Clean up

I. Protocol for Plasmid Purification

Using NUNC™ Brand DeepWell Plates
• Featuring NUNC Glass Fiber Filter Plates (Binding Plates), Cat. No. 278010
Bacterial culture and harvest:
1. Grow bacteria in 1.5 ml of growth medium containing the appropriate antibiotics in a NUNC
2ml DeepWell Plate (sterile) for 16-20 hours at 37°C. Cover Plate with NUNC lid or rayon tape.
2. Harvest the bacteria by centrifugation (2000 x g) 5 minutes.
This should provide ample DNA for downstream use. If more plasmid DNA is desired, incubate
the same bacterial culture in several culture plates (up to 4 plates).

Alkaline lysis:
1. Re-suspend the bacteria pellets in 100 µl of GTE solution, and incubate at room temperature
for 5 minutes.
2. Add 200 µl of lysis solution and seal the plate with NUNC polyester sealing tape. Invert 6-10
times. Incubate at room temperature for 3-5 minutes (not over 5 minutes).
3. Add 200 µl of neutralization buffer, mix well and incubate for 5 minutes on ice.
4. Add 500 µl 6 M guanidine hydrochloride and mix well.

Lysate clarification and DNA binding:

1. Load the lysate into NUNC 1 ml Fritted Filter Plate (lysate clarification plate).
2. Vacuum the clarification plate (-10 in. to -15 in. Hg) and collect the filtrate into NUNC 1 ml
Glass Fiber Filter Plate (binding plate).
3. Vacuum the binding plate with low vacuum pressure (-5 in. to -10 in. Hg), until all solution has
passed through the membrane. Discard the filtrate.
4. Repeat steps 1-3, if lysate from several bacteria culture plates is combined for increased DNA
amounts.
5. Vacuum the binding plate with high vacuum pressure (-15 in. to -20 in. Hg) for 2 minutes.

Wash and elution:

1. Add 1 ml of wash buffer into each well of the binding plate. Vacuum at low pressure (-5 in. to
-10 in. Hg) until all of the solution passes through the membrane. Repeat one time.
2. Vacuum the binding plate with high vacuum pressure (-15 in. to -20 in. Hg) for 1 minute, and
remove the waste container.
3. Dry the membrane in the binding plate under vacuum (-15 in. to -20 in. Hg) for 5 minutes.
4. Add 50-150 µl of elution buffer directly to the center of membrane, and dissolve DNA for 5
minutes at room temperature.
5. Vacuum (-15 in. to -20 in. Hg, for 2 minutes) the binding plate and collect elute into a NUNC
1 ml DeepWell Plate.
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 6. Elute any residual DNA by adding an additional 50-150 µl of elution buffer directly into center of
membrane.
7. Stack Glass Fiber Filter Plate over the same 1 ml DeepWell Plate used in step 5, and
centrifuge (2000 x g) 5 minutes. The purified plasmid DNA in the reception plate is ready for
further work.

Solutions
• GTE Solution - 50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA, 100 µg/ml RNase A, pH 8.0
• Lysis Solution - 0.2 N NaOH, 1% SDS
• Neutralization Buffer - 5M potassium acetate 60 ml, glacial acetic acid 11.5ml, water 28.5 ml
• Wash Buffer - 10 mM Tris-HCl, 1mM EDTA, 80% ethanol, pH 7.4
• Elution Buffer -10 mM Tris-HCl, 1 mM EDTA, pH 8.5

Related NUNC™ Brand Products

• DNA Plasmid Prep Kit (Five 96-well preps), Cat. No. 266000*
• 1 ml DeepWell Plates, Cat. Nos. 260251 (sterile), 260252 (non-sterile)
• 2 ml DeepWell Plates, Cat. Nos. 278743 (sterile), 278752 (non-sterile)
• 1 ml Glass Fiber Filter Plate (Binding Plate), Cat. No. 278010
• 1 ml Fritted Filter Plate (Lysate Clarification Plate), Cat. No. 278011
• 450 µl Conical MicroWell Plate, Cat. No. 249944 (also perfect for final DNA elution of smaller
volumes).
• Rayon Breathable Sealing Tape, Cat. No. 249720
• Polyester Sealing Tape, Cat. No. 236370
• MicroWell Plate Lid (sterile), Cat. No. 264122
* Excludes buffers and solutions. Easy-to-make formulas provided in protocol.

Other protocols, Tech Notes and Bulletins featuring NUNC products are available on our website:
www.nuncbrand.com.

FOR RESEARCH ONLY

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II.Protocol for PCR Product Clean Up
Using NUNCTM Glass Fiber Filter Plates, Cat. No. 278010
Adjust PCR mix for DNA binding:
After PCR amplification, adjust PCR mix to contain 0.3M potassium, 0.5M acetate and
2M guanidine hydrochloride*. Add 10 mM EDTA (pH 8.0) to adjust volume as per the following
chart:
PCR mix volume 50 µl 100 µl
10 mM EDTA (pH 8.0) 120 µl 70 µl
3M potassium 5M acetate 30 µl 30 µl
6M guanidine hydrochloride 100 µl 100 µl
Total volume 300 µl 300 µl

*For small PCR products (<300bp), the recovery rate can be increased by reducing potassium
concentration to 0.15M and acetate concentration to 0.25M in the PCR Mix and Wash Buffer 1.

DNA binding:
1. Load mix into wells of Nunc glass fiber filter plate.
2. Vacuum the glass fiber filter plate with low vacuum pressure (-5 in. to -10 in. Hg), until all
solution has passed through the filter.
3. Vacuum the plate with high vacuum pressure (-15 in. to -20 in. Hg, for 1 minute) and discard
the filtrate.

Wash:
1. Add 50 µl of wash buffer 1 directly onto center of filters and vacuum at low pressure (-5 in.
to -10 in. Hg), until all solution has passed through the filter*.
2. Add 1 ml of wash buffer 2 into wells of plate and vacuum at low vacuum pressure (-5 in. to
-10 in. Hg), until all solution has passed through the filter. Repeat step 2 once.
3. Vacuum the plate with high vacuum pressure (-15 in. to -20 in. Hg, for 1 minute), and
remove the waste container.
4. Dry the filter in glass fiber filter plate under vacuum (-15 in. to -20 in. Hg, for 5 minutes).

Elution:
1. Add 50 µl of elution buffer directly onto center of filters. Let sit for 5 minutes at room
temperature.
2. Vacuum the plate (-15 in. to -20 in. Hg, for 1 minute) and collect eluate into Nunc 1 ml
deepwell reception plate.
3. Add an additional 50 µl of elution buffer directly onto the center of the filters. Stack glass
fiber filter plate over same deepwell reception plate, and centrifuge (2000 x g, 5 minutes) to
elute any residual PCR product. The cleaned up PCR product in reception plate is ready for
further work.

Solutions:
• 3M potassium 5M acetate - 5M potassium acetate 60 ml, glacial acetic acid 11.5 ml,
water 28.5 ml.
• Wash Buffer 1 - 3M potassium 5M acetate 5ml, 6M guanidine hydrochloride 16.7 ml, add
water to 50 ml. (Final concentration is 0.3M potassium, 0.5M acetate, 2M guanidine
hydrochloride). 3
 • Wash Buffer 2 - 10 mM Tris-HCl, 1 mM EDTA, 80% ethanol (pH 7.4)
• Elution Buffer - 10 mM Tris-HCl, 1 mM EDTA (pH 8.5)

Related NUNCTM Brand Products

• 1ml Deepwell Plates, Cat Nos. 260251 (Sterile), 260252 (Non-sterile)

FOR RESEARCH ONLY

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