Team:Hawaii/Test Competent E. Coli

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Contents

Test Competent E. coli DH5a

  • Test our competent cells against Callahan competent cells

Methods

Materials

  • Transported 50uL of competent cells from Callahan Lab on ice
  • 100uL batch 1 competent cells made at OD600 ~0.6 (split into two 50 uL aliquots 4-18a,4-18b)
  • 100uL batch 2 competent cells made at OD600 ~0.3 (split into two 50 uL aliquots 2-17a,2-17b)

Procedure

  1. Thaw plasmid on ice
  2. Add 1uL plasmid pBluescript
  3. Incubate plasmid+competent cells on ice for 30 minutes
  4. Heat shock 60 sec at 42C
  5. Add 250 μl SOC
  6. Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
    1. For plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, growing for 2 hours yields many more colonies
    2. Ampicillin and kanamycin resistant transformants appear to do fine with 1 hour growth
  7. Plate 20 uL dilutions 1/1, 1/10, 1/100 to test competency


Results


Discussion

Next time, remember to aliquot out and transform only 50 ul of the competent cells.

  • What was learned and how to do future experiments differently.
Insanity is doing the same thing over and over again and expecting different results. - Albert Einstein


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