BioCurious: DIY Ultrasonic Transformation: Difference between revisions
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For an XX μL transformation: | For an XX μL transformation: | ||
Equipment: | Equipment: | ||
Off-the-shelf Ultrasonic Jewellery cleaning box with | |||
Off-the-shelf Ultrasonic Jewellery cleaning box with ~ 1L bath | |||
Strains | Strains | ||
DNA | DNA | ||
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Sonicator Essentials: | Sonicator Essentials: | ||
42 KHz | 42 KHz | ||
35 watt peak | 35 watt peak | ||
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## 72 °C 5 min | ## 72 °C 5 min | ||
## 12 °C hold | ## 12 °C hold | ||
---- | |||
Still in progress, above kept for template. | |||
Sonicate each tube for 10 secs at only available setting on Sonicator (on/off)except 2nd control tube with plasmid and Al | |||
#Test transform Tube 1 with Al placed in middle of Transducer bump, flat glass bottom down | |||
#Test transform Tube 2 without Al placed in middle of Transducer bump same way | |||
#Test transform Tube 3 with Al placed in a side of the bath away from transducer | |||
#Test transform Tube 4 without Al placed in a side of the bath away from transducer | |||
#Control Tube 1 without plasmid and Al placed in middle of transducer bump | |||
#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 LB/AmpC after 1 hour and incubate for a day | |||
##Check plates for colony in UV light next day | |||
==Notes== | ==Notes== | ||
Latest revision as of 19:35, 18 March 2013
Overview
Experiment: Transform HB101 Strain with pGlo plasmid using ultrasound device
Materials
For an XX μL transformation:
Equipment:
Off-the-shelf Ultrasonic Jewellery cleaning box with ~ 1L bath Strains DNA reagent in Dark Glass flat bottom tubes 5 mL with Plastic Caps
Sonicator Essentials:
42 KHz 35 watt peak 20 sq cm transducer area in center of bath Bath filled with tap water at room temperature
Procedure
- In a PCR tube, mix the components on ice in the order they are listed above.
- Perform thermocycling program
- 95 °C 5 min
- 95 °C 30 s
- TH 30 s
- 72 °C 1 min for each 1 kb PCR product
- Repeat steps 2-4 a total of 12-36 times (24 is standard).
- 72 °C 5 min
- 12 °C hold
Still in progress, above kept for template.
Sonicate each tube for 10 secs at only available setting on Sonicator (on/off)except 2nd control tube with plasmid and Al
- Test transform Tube 1 with Al placed in middle of Transducer bump, flat glass bottom down
- Test transform Tube 2 without Al placed in middle of Transducer bump same way
- Test transform Tube 3 with Al placed in a side of the bath away from transducer
- Test transform Tube 4 without Al placed in a side of the bath away from transducer
- Control Tube 1 without plasmid and Al placed in middle of transducer bump
- 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 LB/AmpC after 1 hour and incubate for a day
- Check plates for colony in UV light next day
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Relevant papers and books
- Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
- Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
- Ptashne, M (2004) Genetic Switch: Phage Lambda Revisited - Cold Spring Harbor Laboratory Press ISBN 0879697164
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.
