PCR Purification Kit

Supporting Data

Figure 1. High Yield Purification. The high yield of Norgen's PCR Purification Kit is illustrated by purifying 3.5 µg of a 500 bp PCR-amplified product from a reaction mixture and comparing the recovery with a competitor. The purifications were performed in triplicate. DNA recovery was quantified by running 350 ng (1/10th) of the input and 5 µL (1/10th) of 50 µL DNA elution on a 1X TAE, 1% agarose gel followed by densitometry. The average yield for each kit is shown in Figure 1. Norgen's kit shows a significantly higher recovery than the competitor's kit.

Figure 2. Removal of Primer-Dimers. Primer-dimers present in PCR reactions are effectively removed during the purification process with Norgen's PCR Purification Kit. One microgram of a 700 bp PCR fragment, spiked with 250 pmole of primer (Lane 1) was purified using Norgen's kit, with high recovery and no traces of spiked primer present in the elutions (Lanes 2 and 3). Lane 1 contains 400 ng (40%) of the input, and Lanes 2 and 3 contain 20 µL (40%) of the 50 µL elution (duplicate). Lane M is Norgen's MidRanger 1kb DNA Ladder. Eluted DNA was resolved on a 1X TAE, 1% agarose gel.

Kit Specifications - Spin Column
Column Binding Capacity	10 μg
Size of DNA Purified	100 - 15,000 bp
Average Recovery	> 90%
Average Primer Removal	> 90%
Minimum Elution Volume	30 μL
Time to Complete 10 Purifications	15 minutes

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 1 year after the date of shipment.

Component	Cat. 14400 (50 preps)	Cat. 45700 (250 preps)	Cat. 24800 (192 preps)
Binding Buffer C	30 mL	5 x 30 mL	3 x 30 mL
Wash Solution A	12 mL	2 x 20 mL	2 x 38 mL
Elution Buffer B	8 mL	2 x 30 mL	2 x 15 mL
Spin Columns	50	250	-
Collection Tubes	50	250	-
96-Well Plate	-	-	2
Adhesive Tape	-	-	4
96-Well Collection Plate	-	-	2
Elution Tubes (1.7 mL)	50	250	-
96-Well Elution Plate	-	-	2
Product Insert	1	1	2

Documentation

FAQs

Spin Column, High Throughput

Why do I have poor DNA recovery?

Poor DNA recovery could be due to one or a combination of the following factors:

Binding of DNA to the column/wells was inefficient.
Binding of the DNA is dependent on both pH and salt concentration. Ensure that an appropriate amount of Binding Buffer C was used for the volume of the PCR reaction.
The appropriate amount of ethanol was not added to the Wash Concentrate.
The Wash Solution A has been specifically designed to contain the appropriate amount of components. Ensure that the Wash Solution A was prepared using the correct amount of ethanol.
Binding Buffer C was not completely removed in the wash step.
Traces of salt left on the column/wells from the binding step may interfere with the elution of the DNA. Ensure that the columns/wells are washed with the Wash Solution A.
Proper Elution Buffer was not used.
The provided Elution Buffer B has been optimized for high elution recoveries. If water or TE buffer is used instead, ensure the pH is around 8.
Elution Buffer B was not placed directly onto the column bed.
It is important that the Elution Buffer B be placed directly onto the column bed, as this helps to increase recovery by ensuring an even passing of the buffer through the column. Do not pipette the Elution Buffer B onto the side of the column.

Why is the purified DNA not performing well in downstream applications?

Purified DNA may not be performing well in downstream applications due to:

Insufficient washing of plate with bound DNA.
Traces of salt from the binding step may remain in the sample if the columns/wells are not properly washed with the Wash Solution A. Ensure that the columns/wells are washed with the Wash Solution A for 2 minutes during the washing step. Salt may interfere with downstream applications, and thus must be washed from the column.
Eluted DNA contains residual ethanol.
Ensure that the drying step is performed after the second DNA wash to remove all traces of ethanol.

I noticed that there are clogged wells in the plate while using the high throuput kit. Why might this be happening?

Well clogging could be due to the following reasons:

Insufficient Vacuum.
Ensure that a vacuum pressure of at least -650 mbar or -25 in. Hg is developed.
Centrifuge temperature is too low.
Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 15°C may cause precipitates to form that can cause the wells to clog.