Shotgun Library
This protocol is intended to make shotgun libraries from BACS that are to be fully sequenced and assembled. To minimize chimeric clones, an adaptor method is used. There are simpler protocols if just random shotgun sequence is desired.
I. Shearing BAC DNA To shear the large ｉｎｓｅｒｔ ｉｎｓｅｒｔ BAC clone, use the Hydroshear by Genemachines. The settings will vary depending on which orifice you are using, and what size fragments you are trying to generate. New orifices need to be "calibrated" to determine proper settings. The standard we use, to generate predominately 2-5kb fragments is: Cycles: 20 Speed code:10-12.
To make sure BAC DNA is in solution, incubate DNA at 37℃ for 30 minutes, vortexing hard every 10 min. Centrifuge at 12,000 rpm for 4 minutes to determine if the DNA is completely in solution. The DNA is not in solution if a clear pellet can be observed. Transfer the top 190µl to a clean 1.5ml microfuge tube, and proceed with shearing. *Note: We have found that the following works well, when BACDNA is isolated from 500mls LB culture, using the Qiagen-500 tips, and resuspending in 600µl T10E1 at the end. Depending on how concentrated the DNA looks on a gel, dilute an aliquot in water. 100µl of BAC DNA diluted in 100µl water has worked well in the past.
Shearing volume should not exceed 200µl
Wash shearing orifices before and after each sample with the following settings:
*All solutions must be filterd with a 0.2 micron filter before using.
0.2 M HCl x2
0.2 M NaOH x3
ddH2O x5
Please use the Hydroshear manual pgs. 45-46 for instructions to shear DNA.
**Important: Before shearing, DNA must be in solution, or the Hydroshear will clog.
II. DNA Precipitation
After shearing, precipitate DNA with 1/10 volume 3M NaOAc, and 2 volumes ethanol. Spin and wash with 70% ethanol. Dry pellet and resuspend in 150µl water.
III.Repairing ends of the sheared DNA
Add the following components to a microfuge tube:
| Sheared DNA (10-50g)dH2OdNTPs (25mM mixture of 4 dNTPs)BSA (10mg/ml)10X T4 DNA Polymerase BufferT4 DNA PolymeraseTotal Vol. | 150.0µl23.5µl1.5µl, final conc. = 125µM1.0µl, final conc. = 50mg/m20.0µl4.0µl, 12 units200µl |
Incubate at 16℃ for 1 hour.
Incubate at 65℃ for 5 min. to inactivate the enzyme.
Precipitate the BAC DNA with 1/10 volume 3M NaOAc pH 5.2 and 2 volumes 100% EtOH.
Sit on ice for 15 minutes
Centrifuge for 30 minutes at 14,000rpm.
Carefully wash pellet with 1.0ml 70% EtOH. Centrifuge 5 min.
Dry time for pellet is usually 5-10 minutes.
*Note, it's important to not let the pellet over dry, or it will be difficult to get into solution!
相关文章
