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
- 700µl cell broth (overnight gorwth)
- 300µl 50% Glycerol
Store at -80ºC
Miniprep pUA66katE
I the proceeded to miniprep the overnight growth
- 500µl P1, resuspension
- 250µl P2, lysis (wait 4minutes)
- 350µl N3, neutralisation
- Centrifuge at 13,000 rpm for 10minutes
- Move supernatant to Quick Column
- Centrifuge at 13,000 rpm for 1 minute, discard flow-through
- 500µl PB Buffer
- Centrifuge at 13,000 rpm for 1 minute, discard flow-through
- 750µl PE Buffer (with ethanol)
- Centrifuge at 13,000 rpm for 1 minute, discard flow-through
- Centrifuge at 13,000 rpm for 1 minute, discard flow-through
- Place column in eppendorf tube
- 30µl H20, wait for 1 minute
- 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.
- 0.5µl Ligase
- 1µl 10xLigase Buffer
- 2µl pSense66 (2300bp)
- 3µl pBR322 (790bp)
- 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
- Lane 1: 10µl 1kb NEB Quick Ladder
- Lane 2: BLANK
- Lane 3: 50µl pcr product expected 2.3kb (template pUA66katE)
- Lane 4: BLANK
- Lane 5: 50µl pcr product expected 2.3kb (template pUA66katE)
- Lane 6-8: BLANK
Error: No product found
Gel Picture pBR322, 2% Gel
- Lane 1: 10µl 100bp NEB Quick Ladder
- Lane 2: BLANK
- Lane 3: 50µl pcr product, pBR322, expected 790bp (template pMAK512)
- Lane 4: BLANK
- Lane 5: 50µl pcr product, pBR322, expected 790bp (template pMAK512)
- 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
- 80.2µl H20
- 8µl 10xpfu Polymerase Buffer
- 2µl pUA66katE plasmid template (20ng/µl)
- 2.5µl Primer forward
- 2.5µl Primer reverse
For each reaction add the following to each 50µl PCR tube
- 47.6µl Mastermix
- 0.4µl 10mM dNTP (not supplied with kit)
- 1µl pfu Polymerase
PCR conditions
Cycles: 30x
Lid: 100ºC
Volume: 50µl (each)
- Initial: 94ºC, 1 min
- Denature: 94ºC, 30 sec
- Annealing (Tm): 56ºC, 50sec (Lowest Tm 66ºC-10ºC)
- Extension: 72ºC, 4min 43sec (2 min/kb x 2.359kb)
- Goto 2, 30 times
- Final: 72ºC, 10 min
- Rest: 8ºC, forever
Transformation of ligation
I setup a transformation of my ligation on a Kanamycin plate and using heat-shock
- Place 1 tube of 50µl competent cells on ice for 5minutes
- Add 10µl Ligase reaction (or the whole reaction) whilst on ice
- Wait 5 minutes
- Place in 42ºC water bath for 1 minute
- Place back on ice for 5 minites
- Add 250µl prewarmed SOC
- Incubate in shaker for 1 hour
- Spread on plate using glassbeads
Incubate overnight at 37ºC
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