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



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



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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