Greetings All,
I need help working with BACs. I need DNA from 13 BACs for use with the Nextera XT DNA kit to be sequenced on the Illumina MiSeq. I am totally totally new to working with BACs. I've worked with RNA and prepped cDNA libraries with TruSeq kit and DIY, so I understand the need for high quality starting product. The BACs were purchased, I streaked them out on plates, and then followed the miniprep protocol that I was given (see below).
I have been told (and seen on a gel) that there is a ton of RNA in a BAC miniprep. Another source indicated this was a "dirty" prep. What steps should I take to clean this DNA? Should I start over and use a different miniprep protocol? All advice is greatly appreciated!
Thanks,
Melanie
BAC Miniprep:
1. Pick single BAC colony and inoculate a 13mm x 100mm tube containing 2ml Terrific Broth+25ug/ml chloramphenicol.
2. Grow overnight (~16hrs) at 37 C 225-250rpm.
3. Collect cells by centrifugation in micofuge tube at max speed for 40sec. Remove supernatant.
4. Resuspend cells in 400ul GTE by pipetting up and down.
5. Add 400ul FRESH NaOH-SDS. Mix by very gentle inversion ONE time. (Do no more than 12 at a time to limit the lysis time.) The go immediately to next step.
6. Add 400ul KOAc. Mix by inversion, only, and incubate on ice 10min.
7. Centrifuge 8min. max. speed.
8. Transfer superntn’t to new tube by pouring.
(Optional: Centrifuge at max speed 5 min. Pour supernatant into another new tube.)
9. Add 0.6 vol. isopropanol, mix by gentle inversion 15 times.
10. Centrifuge max speed 7 min.
11. Pour off superntn’t. Wash pellet with 1.5ml 70% EtOH, let sit on bench 15 min.
12. Pour off liquid, then add another 0.5ml 70% EtOH, pour off.
13. Centrifuge 1 min max and take off ALL the liquid with a pipettor.
14. Place tubes in rack with tops open and let them dry at 37 C for about 20 min or until pellet becomes transparent.
15. Add 60ul TE and let sit 2h at RT or O/N 4 C.
I need help working with BACs. I need DNA from 13 BACs for use with the Nextera XT DNA kit to be sequenced on the Illumina MiSeq. I am totally totally new to working with BACs. I've worked with RNA and prepped cDNA libraries with TruSeq kit and DIY, so I understand the need for high quality starting product. The BACs were purchased, I streaked them out on plates, and then followed the miniprep protocol that I was given (see below).
I have been told (and seen on a gel) that there is a ton of RNA in a BAC miniprep. Another source indicated this was a "dirty" prep. What steps should I take to clean this DNA? Should I start over and use a different miniprep protocol? All advice is greatly appreciated!
Thanks,
Melanie
BAC Miniprep:
1. Pick single BAC colony and inoculate a 13mm x 100mm tube containing 2ml Terrific Broth+25ug/ml chloramphenicol.
2. Grow overnight (~16hrs) at 37 C 225-250rpm.
3. Collect cells by centrifugation in micofuge tube at max speed for 40sec. Remove supernatant.
4. Resuspend cells in 400ul GTE by pipetting up and down.
5. Add 400ul FRESH NaOH-SDS. Mix by very gentle inversion ONE time. (Do no more than 12 at a time to limit the lysis time.) The go immediately to next step.
6. Add 400ul KOAc. Mix by inversion, only, and incubate on ice 10min.
7. Centrifuge 8min. max. speed.
8. Transfer superntn’t to new tube by pouring.
(Optional: Centrifuge at max speed 5 min. Pour supernatant into another new tube.)
9. Add 0.6 vol. isopropanol, mix by gentle inversion 15 times.
10. Centrifuge max speed 7 min.
11. Pour off superntn’t. Wash pellet with 1.5ml 70% EtOH, let sit on bench 15 min.
12. Pour off liquid, then add another 0.5ml 70% EtOH, pour off.
13. Centrifuge 1 min max and take off ALL the liquid with a pipettor.
14. Place tubes in rack with tops open and let them dry at 37 C for about 20 min or until pellet becomes transparent.
15. Add 60ul TE and let sit 2h at RT or O/N 4 C.
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