Re: [DIYbio] Re: Glass bead transformation

Constanzo and Fox5 demonstrated that intact S. cerevisiae cells can be transformed by agitation with glass beads in the presence of carrier DNA and plasmid DNA at very low efficiency, namely about 300 transformants/µg of plasmid DNA. Osmotic support (with 1.0 M sorbitol) is required in the selective solid medium. (from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056089/)

Sounds good

300 transformants would be great....

On Tue, Aug 5, 2014 at 6:09 PM, Mega [Andreas Stuermer] <masterstorm123@gmail.com> wrote:

It seems pretty convenient. In the book they didn't make protoplasts at first. It seemed to penetrate cell wall anyway.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768579/

"Glass bead transformation of Gram positive bacteria was conducted as follows. Aliquots of 0.5 ml of protoplast suspension were placed in 15 ml conical disposable polypropylene centrifuge tubes (Corning, USA). One µg of pGK12 was added to the protoplasts, followed immediately by the addition of 500 µl of 30% polyethylene glycol 6,000 (PEG 6,000) and 0.3 g of acid washed glass beads (212-300 µm in diameter, Sigma, USA). Protoplasts were agitated at the highest speed on a vortex mixer for 15 sec, and then diluted by the addition of 10 ml of transformation buffer. After the beads were allowed to settle, agitated protoplast suspension was transferred to a new 15 ml conical tube. Cells were pelleted by centrifugation at 5,000 xg for 5 min and suspended in 1 ml of BHI supplemented with 0.5 M sorbitol. After incubation at 37°C for 1 h, transformants were recovered by plating the protoplast culture onto BHI agar containing 0.5 M sorbitol and erythromycin at the final concentration of 3 µg/ml and incubated at 37°C for 5 to 7 days" 

--
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diybio@googlegroups.com. To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---

You received this message because you are subscribed to the Google Groups "DIYbio" group.
To unsubscribe from this group and stop receiving emails from it, send an email to diybio+unsubscribe@googlegroups.com.

To post to this group, send email to diybio@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio.

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/cc5a7d8a-e5ab-4340-a35a-88c100001be6%40googlegroups.com.

For more options, visit https://groups.google.com/d/optout.

--
----------------------------------------
Brian Degger

twitter: @drbrian

http://makerspace.org.uk

http://transitlab.org

----------------------------------------

--
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diybio@googlegroups.com. To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to a topic in the Google Groups "DIYbio" group.
To unsubscribe from this topic, visit https://groups.google.com/d/topic/diybio/RozRlR6ccqQ/unsubscribe.
To unsubscribe from this group and all its topics, send an email to diybio+unsubscribe@googlegroups.com.
To post to this group, send email to diybio@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio.

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/CACc%3DpRLseKhm17HCdd-8snzpXT-2gZRuQBqW_utEvjxcavt-bw%40mail.gmail.com.

For more options, visit https://groups.google.com/d/optout.