IGEM:IMPERIAL/2006/LabCalendar/2006-7-27

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Maxiprep

  • Transfer maxiprep cultures that were in the 500 mL flasks to 50 mL falcon tubes
  • Centrifuge for 5 minutes at 3000 rpm
  • Follow instructions for maxiprep on kit
  • Part 6B filter broke, but still managed to obtain some DNA

Finding the concentration of DNA after maxiprep

  • Use a quartz cuvette
  • Set at 260 nm
  • Calibrate with 1 mL distilled water
  • Add in 5 uL of DNA sample
  • Can repeat for another 3-4 times before rinsing it out, just calibrate it

Results

  • 2H - 0.011 % ABS
  • 6B - 0.008 % ABS
  • 9G - 0.007 % ABS
  • 12D - 0.008 % ABS
  • 7A - 0.003 % ABS (?) Could not get an accurate result
  • 24A - 0.011 % ABS
  • 3O - 0.008 % ABS


Preparing the digests to extract the DNA from the gels (30 uL digests)

  • 1 uL of each enzyme
  • 25 uL DNA sample
  • 3 uL proper buffer

Run Gels

  • Since they are the large plates, run at 130 V and 0.13 A
  • Let them run for approximately 1 hour

Extract DNA from gels

  • Ensure you know which line to cut out
  • Place gel on the transilluminator
  • Cut out part using scalpel

Parts cut out

  • 7A - 756 BP part
  • 24A - 2740 BP part
  • 12D - 875 BP part
  • 3O - 2091 BP part
  • 2H - 3381 BP part

These were easily visible in pink on the transilluminator and were cut out and placed into an eppendorf tube

Follow the protocol for extracting the DNA (Glass Milk DNA Purification)

  • For the inserts, use 20 uL in step 10, but for vectors use 100 uL water
  • In step 12, 16 uL for the insert to be transferred into labelled tube, but 50 uL for the vector into labelled tube

DNA ligation

  • 1 uL vector (keep remaining vector sample in fridge)
  • 2 uL ligase buffer (found in freezer)
  • 1 uL ligase (also in freezer)
  • Keep mixture at room temperature overnight and allow to ligate

Parts ligated:

  • 12D into vector 24A (I13504 into vector C0261)
  • 12D into vector 2H (I13504 into vector I13033)
  • 7A into vector 3O (C0062 into vector B0034)

To do tomorrow:

  • Electroporate ligated parts into DH5a
  • Also electroporate non-cultured part into DH5a from parts registry (terminator B0015)
  • Grow on plates
  • Miniprep parts 2H (I13033), 16P (J04500), 5M (C0060) again. First attempt at miniprep failed to show any bands upon digesting and running on gel
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