Re: [DIYbio] Re: cheaper/better transformation - using ultrasound (sonoporation)

Hi Guys,
     Per Patrik's request...
    Kyle Taylor,Jay Hanson and self  performed the experiment here at Biocurious, initial results are encouraging..
Only deviations from paper were because of material at hand availibility and additional Al foil cutting in tube..

-shashank


Experiment: Transform HB101 Strain with pGlo plasmid using Ultrasound device

Equipment:   Off the shelf Ultrasonic Jewellery cleaning box with about 1l bath.
              Strains, DNA and reagent in Dark Glass flat bottom tubes 5ml with Plastic Caps

Sonicator Essentials:
        42 KHz
        35 watt Peak
        20cm sq transducer area in center of bath
                      Bath filled with Tap water at room temperature
Transform Tubes:
    Four Tranform tubes with

1. 500ul of  O.N. culture of HB101 strain
2. 5ul of pGlo plasmid prepared thru miniprep
3. 1ml of 75mM CaCl2
4. Two tubes with Al foil discs (two none)

    Two Control Tubes

1.    Same as above , but no plasmid, no Al
2.    Same as Transform, with plasmid and Al

Procedure:
      Each Tube was sonicated for 10 secs at only available setting on Sonicator (on/off)
      except 2nd control tube with plasmid and Al

1.       Test transform Tube 1 with Al placed in middle of Transducer bump, flat glass  bottom down
2.      Test transform Tube 2 without Al placed in middle of Transducer bump same way
3.      Test transform  Tube 3 with Al placed in a side of the bath away from transducer
4.      Test transform  Tube 4 without Al placed in a side of the bath away from transducer
5.      Control  Tube 1 without plasmid and Al placed in middle of transducer bump
6.      Control Tube 2 with plasmid and Al left alone

   
    Post sonication
        Add 1 ml LB solution
        incubate at 37C 1 hr on a bath-shaker
                      Plate on LB and Amp agar after hour and incubate for a day
                      Check plates for colony in UV light next day



Results:
    Test tube

1. With HB/PGlo/Al/midTransducer: 9 Colonies
2. with HB/PGlo/midtransducer: 4 colonies
3. with HB /PGlo/Al/side placed: 3 colonies
4. with HB/Pglo/side placed: 1 colony

     Control
    1. Just bacteria/midplaced: 0 colony
           2. Bacteria/Pglo/Al no sonication: 0 colony

FWIW, we are pretty happy about it...open to all ideas to improve it.


On Friday, March 1, 2013 4:28:35 AM UTC-8, Patrik D'haeseleer wrote:

Hold yer horses! I wasn't involved in this experiment - just forwarded the picture. I'll let you know as soon as I know any more details.

Yes, the yield looks low, but I doubt they did much (or any?) optimization of the protocol so far. Not too bad for something that worked on the first try!

Patrik

On Friday, March 1, 2013 3:23:57 AM UTC-8, Avery wrote:

well done!  I am also curious.  That looks like pretty low yield for compared to what i normally see from heat shock, but I would like to know more about the protocol.

--A

On Fri, Mar 1, 2013 at 4:42 AM, Mega <masters...@gmail.com> wrote:

Awesome!! How much ug plasmid  did you use?

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