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origami bacteria transformation


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#1 marzieh

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Posted 08 November 2013 - 12:17 AM

Hello. I am trying to transform Origami B (DE3) host cells but I find lots of problems to grow and transform the cells. I used cacl2 to make them competent.The cells didn't grow on the selection plate after transformationunsure.png . (Using the same protocol, BL21 cells have been transformed efficiently).



#2 Missle

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Posted 08 November 2013 - 05:09 AM

I didn't know that you could buy Origami cells that weren't already competent?



#3 bob1

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Posted 08 November 2013 - 03:17 PM

So - what are the problems?  How did you grow the cells?  How did you transform them?  Details are the key to troubleshooting.



#4 marzieh

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Posted 09 November 2013 - 05:30 AM

Thanks for your answers. I used standard CaCl2/heat shock method for preparation  and transformation of competent cells. After transformation, approximately 100 ul  of transformed competent cells were transferred onto agar lB medium and incubated at 37°C.No colony was appeared after 24 hours.(origami cells grow slowly)


Edited by marzieh, 09 November 2013 - 06:34 AM.


#5 marzieh

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Posted 09 November 2013 - 06:33 AM

I followed all the protocol recommendations, so i assume that the failure is due to the cell strain itself (considering that the BL21 cells were transformed efficiently)

my detailed protocol:

1.10 ml LB was inoculated with 2 ml of fresh overnight (ON) culture and  incubated for 2.5 hours at 37° 

2.OD600 was reached to 0.4-0.6

3.the culture was incubated on ice bath for 10 min

4.the cells were recovered by centrifugation at 4100 rpm  for 10 min at 4°C

5.pellet was resuspended in 2 ml of ice-cold cacl2 (100mM)

6.the suspension was incubatedon ice bath for 20 min.

7.step 4 was repeated

8.pellet was resuspended in 400 ul of ice-cold cacl2 (100mM) plus 120 ul of glycerole

9.the cells were stored  in -80°C in 100 ul aliquots.

(transformation with 20 ul of target plasmid was carried out after 2 days)



#6 phage434

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Posted 09 November 2013 - 07:26 AM

I'd suggest that rather than guessing, you should measure the competence of your cells. Likely it is very poor, with this protocol. I'd suggest doing it with BL21 (DE3) cells in parallel, so that you can compare with cells you know work. You measure competence by transforming with serial dilutions of a known working plasmid, finally determining colony forming units per microgram of plasmid. For BL21 strains, something in the 10^5 CFU/ug is a reasonable number. If necessary, you can try a higher efficiency competent cell protocol.

 

Are you paying attention to the antibiotic resistances of these strains? Some of the unusual BL21 derivatives have plasmids already with chloramphenicol resistance (Rosetta) or chrlomosomal integrations carrying resistances kanamycin and tetracycline (Origami 2 (DE3). So, if you were trying to transform a plasmid with Kan resistance, all the cells would grow, and you perhaps would see the lawn and think that no colonies formed. Always check the genotype. This is easy to spot -- streak a plate rather than spread it.






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