Jun 13, 2023
Size selection (Purification)
Forked from Size selection (Purification)
- 1Kaohsiung Medical University
Protocol Citation: Tsu-Chun Hung, Yin-Tse Huang 2023. Size selection (Purification). protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9pezzg3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 13, 2023
Last Modified: September 13, 2023
Protocol Integer ID: 83294
Abstract
Size selection (Purification)
Calculate the amount of magnetic beads you need and DISTRIBUTE in a new 1.5 mL eppendorf tube (Mix the bottle VIGOROUSLY to make sure it's homogeneous).
For example, if you have 5 samples to clean up, distribute 49.5 μl in a new 1.5 mL eppendorf tube.
| A | B | C | |
| Sample | Magnetic beads need | Magnetic beads need *1.1 | |
| 1 | 9 μl | 9.9 μl | |
| 2 | 18 μl | 19.8 μl | |
| 3 | 27 μl | 29.7 μl | |
| 4 | 36 μl | 39.6 μl | |
| 5 | 45 μl | 49.5 μl | |
| 6 | 54 μl | 59.4 μl | |
| 7 | 63 μl | 69.3 μl | |
| 8 | 72 μl | 79.2 μl |
Note
The ratio is 20μl of sample use 9μl of magnetic beads to clean up (0.45X to keep long DNA fragments).
Note
Magnetic bead: BeaverBeads™ DNA Select Isolation
In each 20 µL sample, add 9 µL magnetic beads in.
Note
Pipetting thoroughly every time before adding to make sure all magnetic beads mixed well.
Mix sample and beads by gently flicking the tube for 5mins 00:05:00
Note
Be gentle, mix too aggressive will make DNA fragmented
5m
Transfer the tube to the 8-strip magnetic rack.
After all the magnetic beads are arrested, remove the supernatant.
Add 200 µL 80% ethanol, flip the magnetic rack around to clean the pellet.
Wait until all the magnetic beads are arrested, remove the supernatant.
Note
FRESHLY make 80% ethanol
Repeat the step 5.
Flash spin the tube, and put on the magnetic rack.
Remove ANY trace of liquid residuals using 10 μl pipette.
Note
Caution: DO NOT let the beads crack
Add 10 µL elution buffer.
Mix gently by flicking the tube and make sure all magnetic beads are dissolved in buffer, then flash spin down the sample.
Incubate the tube in PCR thermal cycler using "37_incubation" program for 10min 00:10:00 .
10m
Transfer the tube to the magnetic rack.
After all the magnetic beads are arrested, collect the 10 µL supernatant to a 200 μl PCR tube or a 8-strip PCR tube.
The clean-up sample is now ready for 2' PCR.