Sequencing reaction magnetic beads -20ml

Sequencing reaction magnetic beads

Specifications: NYMB01-20

1、Product Introduction

This kit, through an optimized magnetic bead system, can specifically bind to all fragments in the Sanger sequencing reaction, but does not bind to raw materials such as dNTP, ddNTP, and enzymes. Magnetic beads can unidirectionally adsorb all the fragments produced by PCR amplification. Then, through 80% ethanol washing, all the remaining raw materials of the reaction are removed, thereby achieving the purpose of purification. The purified product can be directly sequenced by electrophoresis on sequencers such as 3730XL. The signal is strong and uniform, with extremely low background noise. It can completely replace the traditional EDTA/ ethanol precipitation method, facilitating automated operation and reducing human errors.

2、Product Features

3、Precautions

1、After each use, store in a 4℃ refrigerator. Do not place it close to the refrigerator wall, as it may freeze. 

2、This product must not be frozen. Before use, the magnetic beads should be thoroughly mixed with a vibration meter.

4、Operation steps：

1、Self-prepared materials: absorbent paper, sterilized deionized water, silicone pad

2、Instructions：

2.1  Add 5μL of sequencing reaction magnetic beads to each well (thoroughly mix before use), 9μL of sterilized deionized water and 26μL of anhydrous ethanol. Cover with a silicone pad, shake and mix well for 15 seconds, let stand for 2 minutes, shake again for 15 seconds, centrifuge to 1500 RPM and stop.

2.2  Place the PCR plate on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely adsorbed, remove the silicone pad, pour out the liquid, and turn it upside down onto absorbent paper to absorb the remaining liquid. If conditions permit, perform an inverted centrifugation at 550rpm for 10 seconds.

2.3  Place the PCR plate still on the magnetic rack, add 100μL of 80% alcohol to each well, let it stand for 1 minute, discard the liquid, turn it upside down on a new absorbent paper, and absorb the remaining liquid. If conditions permit, perform an inverted centrifugation at 550rpm for 10 seconds, replace it with new absorbent paper, and then perform another inverted centrifugation for 30 seconds. 

2.4  Add 60μL of sterilized deionized water to each well, cover with a clean silicone pad, vortex for 30 seconds, and centrifuge at 4000rpm for 3 minutes.

2.5  Place the 96-well plate on a magnetic rack and let it stand for 1 minute. When the magnetic beads are completely adsorbed, carefully transfer the DNA solution to a new 96-well plate for on-machine operation or storage under appropriate conditions.

Peak diagram of bacterial liquid purification

Peak diagram of rubber cutting and purification

Common Problems and Solutions

1、The appearance of interference peaks

1. The magnetic beads were not thoroughly mixed when added. After adding the magnetic beads and ethanol, please mix thoroughly. 

2. After the third step, if the ethanol has not completely evaporated, the remaining ethanol will affect the sequencing electrophoresis. Please make sure the ethanol has completely evaporated.

3. The concentration of the sequencing reaction is very low. This situation mainly concerns BAC terminal sequencing, as the signal value of BAC terminal sequencing is relatively low. Please increase the amount of BigDye used during the reaction process.

2、There is no signal in sequencing electrophoresis

1. Please be sure to elute with deionized water. 

2. The sequencing reaction itself has no signal.

3. The capillary failed to draw the sample. Since deionized water was used for elution, it is relatively volatile. After continuous multi-plate electrophoresis, due to the environment, in a relatively dry environment, the volume of the PCR plate after evaporation is less than 10μl, which causes the capillary to be unable to adsorb the sample during the electrophoresis process. According to the specific situation, the amount of transferred sample can be appropriately increased.