Thank you😀
I got made my own protocol as a spin-off of several. Streak cells onto LB overnight, grow single colony in 15mL LB overnight, take 1mL of overnight culture and Grow cells to OD=0.5 in 125mL of LB shaken for 4hrs-ish (NEB Turbos are fast!) at 37C 200rpm 20mm orbit in a 250mL erlenmeyer flask with foam plug, spin down at 3500rpm for 5 min in 8 15mL tubes full up. Decant completely, remove all the excess liquid with a pipette (very important to do so but do not disturb the pellet). Resuspend one of the 8 tubes with 1mL cold LB, take the entire resuspension into another spun down tube, use the liquid from the previous resuspension to resuspend the second tube. Keep doing this until all the pellets are resuspended and concentrated into one tube. Resulting suspension should be about 1.2mL. Add that volume of ice cold TSS 2x to the tube and mix gently by pipetting up and down. Dispense 100uL into sterile prechilled pcr tube. Makes about 24 reactions worth. TSS is transfer and storage buffer as per the original article on said buffer. I use lithium chloride instead of magnesium. Annecdotal evidence suggests it works better. Make sure to do all the above actions on ice and work quickly. Keep all tubes in contact with ice while resuspending. Using freshly miniprepped plasmid to test I get hundreds of colonies. I heat shock cells by incubating at 42C for 90 seconds in preheated pcr machine and then ice for 5min. Add 100uL SOC to recover if non-amp plates or tricky ligation, shake in pcr tube at 37C 200rpm in glass or plastic 60mm petri dish bottom so tubes rattle around for no less than 20min but no more than 40min. Plate 100uL per pre-warmed lb agar dish with appropriate antibiotics. Enjoy!Sebastian S. CociobaCEO & FounderNew York Botanics, LLCBlog: ATinyGreenCell.com--Which protocol are you using?--Im just confused as to why people buy competent cells when you can easily make them yourself. Biohackers don't have money but have time so why not get a nice plasmid producing strain and make a TSS buffer based competent cell system. Mine last for 3 weeks in -20C. Works fine. I use a line of NEB turbos I bought like three years ago. Unless you have a -80 the price of comp cells are not worth it at all. Its not magic in those cells, its cell state and buffer used...all which can be obtained by the user. Commercial comp cells are just convenience for labs that absolutely need highest efficiency...though for hacker-spaces and personal labs I see no purpose in massively competent transformations. Its one of those fetishized metrics people rave about but its intrinsically pointless for most cases. After three weeks I still get 40 colonies which is more than enough to screen a ligation or colony pcr. Pardon the rant but I really don't see a point in buying when there are so many good protocols for making them simply.
Sebastian S. CociobaCEO & FounderNew York Botanics, LLCBlog: ATinyGreenCell.com--On Saturday, March 5, 2016 at 8:44:54 AM UTC-8, William Beeson wrote:--for their ultra-high competency cells they have a FAQ (https://www.neb.com/faqs/1/01/01/can-i-store-competent- ) that says after 1 week of storage at -20C the efficiency is decreased by 250x.cells-at-20-deg-c-instead-of- 80-deg-c Nice data point! They say "cells lost 94.5% of TE after only 24 hours of storage at -20°C. Cells lost 98.9% of TE after 2 days, and 99.6% of TE after one week of storage at -20°C" - so that would be 18x after one day, 91x after 2 days, and 250x after 7 days.Anyone know whether electrocompetent cells maintain their competence any better, or whether there are any particular strains that tend to maintain competence longer when stored above -80?PatrikPS: Of course, all this also points out how important shipping conditions are likely to be for competent cells
-- 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 https://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/1d273757-64b0-440d-922f-fd55428917fd%40googlegroups.com.
For more options, visit https://groups.google.com/d/optout.
-- 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 https://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/0A472610-3CA9-40C4-9D65-6764B2799F51%40gmail.com.
For more options, visit https://groups.google.com/d/optout.
-- 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 https://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/E0B830AF-876D-494D-9080-7D7BB6C40A61%40gmail.com.
For more options, visit https://groups.google.com/d/optout.
-- 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 https://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/34AF1FD8-CDC9-4600-B86F-EBFA7CC8AC32%40gmail.com.
For more options, visit https://groups.google.com/d/optout.
0 comments:
Post a Comment