User:Howard Boland/Notebook/Art from Synthetic Biology/2010/10/30

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Check Overnight growth

I checked the overnight growth of two 10ml tubes with pUA66katE. They grew fine.

Glycerol Stock pUA66katE

I made glycerol stock of the overnight growth

  1. 700µl cell broth (overnight gorwth)
  2. 300µl 50% Glycerol

Store at -80ºC

Miniprep pUA66katE

I the proceeded to miniprep the overnight growth

  1. 500µl P1, resuspension
  2. 250µl P2, lysis (wait 4minutes)
  3. 350µl N3, neutralisation
  4. Centrifuge at 13,000 rpm for 10minutes
  5. Move supernatant to Quick Column
  6. Centrifuge at 13,000 rpm for 1 minute, discard flow-through
  7. 500µl PB Buffer
  8. Centrifuge at 13,000 rpm for 1 minute, discard flow-through
  9. 750µl PE Buffer (with ethanol)
  10. Centrifuge at 13,000 rpm for 1 minute, discard flow-through
  11. Centrifuge at 13,000 rpm for 1 minute, discard flow-through
  12. Place column in eppendorf tube
  13. 30µl H20, wait for 1 minute
  14. Centrifuge at 13,000 rpm for 1 minute

Setup 2 hour ligation of pSense66 & pBR322

Both products have been digested, purified and check on gel.

  1. 0.5µl Ligase
  2. 1µl 10xLigase Buffer
  3. 2µl pSense66 (2300bp)
  4. 3µl pBR322 (790bp)
  5. 3.5µl H2O

Incubate at room temperature for 2 hours.

Gel PCR products pSense66 & pBR322

I ran a 1% gel for the pSense66 (2300bp) and a 2% for the pBR322 (790bp).

I found nothing on the pSense66 gel and one band out of two on the pBR322 gel.

Gel Picture pSense66, 1% Gel

  1. Lane 1: 10µl 1kb NEB Quick Ladder
  2. Lane 2: BLANK
  3. Lane 3: 50µl pcr product expected 2.3kb (template pUA66katE)
  4. Lane 4: BLANK
  5. Lane 5: 50µl pcr product expected 2.3kb (template pUA66katE)
  6. Lane 6-8: BLANK

Image:30102010-pcr-2300-porduct-noproduct.jpg

Error: No product found

Gel Picture pBR322, 2% Gel

  1. Lane 1: 10µl 100bp NEB Quick Ladder
  2. Lane 2: BLANK
  3. Lane 3: 50µl pcr product, pBR322, expected 790bp (template pMAK512)
  4. Lane 4: BLANK
  5. Lane 5: 50µl pcr product, pBR322, expected 790bp (template pMAK512)
  6. Lane 6-8: BLANK

Image:30102010-pcr-product-790-gel-cut.jpg

Error: Only one product found

PCR, pSense66 expected 2.3kb

After the unsuccessful PCR of my 2.3kb, I decided to return to my original conditions at the beginning of the week. All conditions appart from the template were used.

Mastermix PCR - pUA66katE

  1. 80.2µl H20
  2. 8µl 10xpfu Polymerase Buffer
  3. 2µl pUA66katE plasmid template (20ng/µl)
  4. 2.5µl Primer forward
  5. 2.5µl Primer reverse


For each reaction add the following to each 50µl PCR tube

  1. 47.6µl Mastermix
  2. 0.4µl 10mM dNTP (not supplied with kit)
  3. 1µl pfu Polymerase


PCR conditions

Cycles: 30x Lid: 100ºC Volume: 50µl (each)

  1. Initial: 94ºC, 1 min
  2. Denature: 94ºC, 30 sec
  3. Annealing (Tm): 56ºC, 50sec (Lowest Tm 66ºC-10ºC)
  4. Extension: 72ºC, 4min 43sec (2 min/kb x 2.359kb)
  5. Goto 2, 30 times
  6. Final: 72ºC, 10 min
  7. Rest: 8ºC, forever

Transformation of ligation

I setup a transformation of my ligation on a Kanamycin plate and using heat-shock

  1. Place 1 tube of 50µl competent cells on ice for 5minutes
  2. Add 10µl Ligase reaction (or the whole reaction) whilst on ice
  3. Wait 5 minutes
  4. Place in 42ºC water bath for 1 minute
  5. Place back on ice for 5 minites
  6. Add 250µl prewarmed SOC
  7. Incubate in shaker for 1 hour
  8. Spread on plate using glassbeads

Incubate overnight at 37ºC


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