APPDE004, APPDE050, APPDE100, APPDE300
α+ SolutionTM
Plasmid Extraction Mini Kit

APPDE 004 4 preps
APPDE 050 50 preps
APPDE 100 100 preps
APPDE 300 300 preps

APPDE 004 APPDE 050 APPDE 100 APPDE 300
PD1 Buffer 1.0 ml 13 ml 25 ml 75 ml
PD2 Buffer 1.0 ml 13 ml 25 ml 75 ml
PD3 Buffer 1.5 ml 18 ml 35 ml 105 ml
PDW Buffer (concentrate) 1.3 ml 23 ml 45 ml 135 ml
Wash Buffer (concentrate) 1.0 ml 10 ml 20 ml 50 ml
Elution Buffer 0.5 ml 7 ml 15 ml 35 ml
PD Column 4 pcs 50 pcs 100 pcs 300 pcs
Collection Tube 4 pcs 50 pcs 100 pcs 300 pcs
RNase A (Lyophilized) 0.15 mg 1.25 mg 2.5 mg 7.5 mg
User Manual 1 1 1 1

 

※ Principle : mini spin column (silica matrix)

※ Sample size : 1 ~ 3 ml

※ Size of plasmid or construct : < 15 kb

※ Operation time : < 25 minutes

※ Typical Yield : 20 ~ 30 μg

※ Binding capacity : 60 μg/ column

※ Column applicability : centrifugation and vaccum

 

Important Notes:

Store RNase A at -20 °C upon recipit of kit.

Add 0.5 ml of PD1 Buffer to a RNase A tube, Dissolve the RNase A by vortexing. Briefly spin the tube and transfer the total RNase A mixture back to the PD1 bottle, mix well by vortexing and store the PD1 buffer at 4 °C.

If precipitates have formed in PD2 Buffer, warm the buffer in 37°C waterbath to dissolve precipitates.

Preparation of PDW Buffer and Wash Buffer by adding 96 ~100% ethanol (not provided) for first use.

Centrifugation steps are done by a microcentrifuge capable of the speed at 11,000 ~1,8000 x g.

Problems

Possible reasons

Solutions

Low yield

Bacterial cells were not lysed completely

Too many bacterial cells were used (OD600 > 10). Separate the bacterial culture into multiple tubes.

 

After PD3 Buffer addition, break up the precipitate by inverting to ensure higher yield.

Overgrown of bacterial cells

Incubation time should not be longer than 16 hours.

Bacterial cells were insufficient

Ensure that bacterial cells have grown to an expected amount (OD600 > 1) after incubation under suitable shaking modes.

Incorrect DNA elution step

Ensure that Elution Buffer was added and absorbed to the center of the PD Column matrix.

Incomplete DNA Elution

If the size of DNA fragments is larger than 10 kb, use a preheated Elution Buffer (60~70°C) on the solution step to improve the elution efficiency.

Incorrect preparation of Wash Buffer

Ensure that the correct volume of ethanol (96 ~ 100 %) was added to Wash Buffer before use.

Eluted DNA does not perform well

Residual ethanol contamination

After Wash Step, dry the PD Column with an additional centrifugation at top speed (~18,000 x g) for 5 minutes or incubation at 60°C for 5 minutes.

Genomic DNA Contamination

Lysate prepared improperly

Gently invert the tube after adding the PD2 Buffer. And the incubation time should not be longer than 5 minutes.

 

Do Not use overgrown bacterial culture.

RNA Contamination Plasmid DNA

Insufficiency of RNase A activity in PD1 Buffer because of long-term storage

Prior to using PD1 Buffer, ensure that RNase A was added. If RNase A added PD1 Buffer is out of date, add additional RNase A into PD1 Buffer to a concentration of 50 μg/ ml then store 4°C.

 

Too many bacterial cells were used, reducing sample volume.

 

Smearing or degrading of Plasmid DNA

Nuclease contamination

If used host cells have high nuclease activity (e.g., enA+ strains), perform the following optional Wash Step to remove residuary nuclease.

 

a. After DNA Binding Step, add 400 μl of PWD Buffer into the PD Column and incubate for 2 minutes at room temperature.

b. Centrifuge at full speed (~18,000 xg) for 30 seconds.

c. Proceed to step 6.2.

Plasmid DNA is not adequate for enzymatic digestions

Eluted plasmid DNA contains residual ethanol

Make sure you have discarded the flow-through after washing with Wash Buffer (Step 6.2) and centrifuged for an additional 3 minutes (Step 7).

Denatured Plasmid DNA migrate faster than supercoiled form during electrophoresis

Incubation in PD2 Buffer too long

Do not incubate the sample longer than 5 minute in PD2 Buffer

 

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