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Old 05-17-2013, 01:46 PM   #1
OnUrMark
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Location: Oregon

Join Date: Jan 2011
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Default How to prep BACs for Nextera XT DNA kit?

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.
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Old 05-20-2013, 08:32 AM   #2
krobison
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A few suggestions:

1) Make sure you are quantifying your DNA by a fluorometric approach (Qubit, PicoGreen, etc) and not just 260/280; 260/280 can be very misleading

2) There is an exonuclease cocktail called PlasmidSafe; use it! Some of the LMW schmutz you are apparently seeing could well be genomic DNA fragments, and these will certainly go into your library and reduce the yield of fragments from your BAC. We've sometimes seen the host background dominate the sequencing run; it can certainly make estimating how much data you need a challenge

3) I wish I understood how sensitive Nextera is to RNA contamination, but make sure you hit the prep hard with RNase.

4) Does your BAC vector contain a conditional origin-of-replication, as many do? You can amplify your BAC yield this way. However, by increasing the copy number you also run the risk of instability; one of the selling points of BACs is the single copy nature.

Good luck!
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Old 05-20-2013, 09:32 AM   #3
OnUrMark
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Hi Krobinson,

Thanks for your advice! I have access to a Qubit for DNA quantitation, and I would like to clean the minipreps I have in hand without amplification.

It sounds like the main thing I need to do is to treat them with RNase and PlasmidSafe. I’m inexperienced with restriction enzyme digestion in general.

1) Would it work to RNase and PlasmidSafe in one step, or should I do it sequentially?

2) If I RNase first, will I need to purify them before treating with PlasmidSafe? (I read that heating does not deactivate RNase.)

3) What type of purification would be best to use after digestion with PlasmidSafe?
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Old 05-20-2013, 12:56 PM   #4
chevrm
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No experience with putting them together but I don't see why not (as long as similar active temps).

Leaving the exo (plas safe) in the sample is fine as long as it's deactivated either by a heat kill or a chelation (EDTA, etc) or both. I'm paranoid and like to double-down. The EDTA would more than likely kill your transposon rxn though, so I'd recommend:

1) RNase + PS Exo w/ heatkill
2) EDTA rxn neutralization (just spike it in, no incubation necessary)
3) QIAquick/SPRI/other purification, elute in Tris or Water (not sure if SPRI recovery would be effected by circ'd DNA, so I'd opt for a column if throughput allows)
4) Nextera........
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