IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/30

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Team Allergy

  • Checked for transformants (seems that only Bet worked but there are only 2 colonies (could be background))
    • Grew up 2 tubes for minipreps at ~10am (Should be ready ~ 4 pm)
  • Miniprepped w/ concetnrations of ~ 80 ng/uL and 108 ng/uL
  • Redid ligations and transformations (plated ~ 12 pm)
    • Bet, LTP, GFP, LTP .5, GFP .5, Negative Control (just V0120 backbone)


Ligation Reactions
Bet GFP LTP LTP(.5) GFP(.5) Negative Control
DNA Insert 1 1 1 1 1 0
T4 DNA ligase buffer (10x) 1 1 1 1 1 1
diH2O 6 6 6 6 6 7
T4 DNA ligase 1 1 1 1 1 1
DNA Backbone 1 1 1 1 1 1
  • Transformed ligations into turbo cells and let it sit outside rather than in 37
  • PCR of PAL3 and PME2 introns from arabidopsis genomic DNA

Team Flavor

Miraculin, Brazzein

  • Colonies did grow!
  • Started 5 mL cultures of Miraculin and Brazzein biobrick constructs (5 cultures each)

The Big Three

  • Began miniprep of cultures of pENT, NOSt+STOP and our Control (for experimental purposes)


OD specs
OD
pENTCUP2 #1 248.5 ng/μL
pENTCUP2 #2 447.7 ng/μL
NosT + stop #1 427.7 ng/μL
NosT + stop #2 303.9 ng/μL
Control #1 366.8 ng/μL
Control #2 359.2 ng/μL


Digestion

  • We digested miniprep samples with xbaI/pstI fast digest enzymes in order to determine if ligation was successful.


Digestion Reactions
Control #1 Control #2 pENTCUP2 #1 pENTCUP2 #2 NosT & stop #1 NosT & stop #2
DNA 2 2 3 2 2 3
Green FD buffer 2 2 2 2 2 2
diH2O 14 14 13 14 14 13
xbaI 1 1 1 1 1 1
pstI 1 1 1 1 1 1


Digestion mixes were placed in 37°C water bath for approximately 1 hr.


Big Three PCR digestion

  • We simultaneously digested PCR purified pENTCUP2, NosT, and NosT & stop inserts with xbaI/pstI. This way if the above digestion showed the ligation of the inserts into v0120 backbone was not successful, we can continue with these inserts.


Digestion Reactions
pENTCUP2 NosT NosT & stop
DNA 4 7 2
Green FD buffer 2 2 2
diH2O 12 9 14
xbaI 1 1 1
pstI 1 1 1


Placed in 37°C water bath for 20 minutes.


Team Fence

pMT1002 Miniprep

  • 4ml from each overnight cell culture of pMT1002 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm
  • remainder of overnight cell cultures were placed in the fridge for transfer to microcentrifuge tubes at a later time
  • excess LP+amp decanted, the pellet resuspended in 250μL P1
  • contents transferred from each 15ml conical to a new epindorf tube
  • 250 μL P2 added to each tube, mixed by inverting gently 4-6 times
  • 350 μL N3 added to each tube, mixed by inverting as before
  • tubes centrifuged for 10 minutes at 13,000 rpm
  • supernatants pipetted into new QIAprep sin columns
  • columns centrifuged for 30-60 seconds, flow through discarded
  • QIAprep columns washed with 0.5 ml buffer PB each and centrifuged for 30-60 seconds
  • columns washed with 0.75 ml buffer PE and again centrifuged for 30-60 seconds
  • flow through was discarded and tubes centrifuged for an additional minute
  • column was placed in a clean 1.5 ml microcentrifuge tube
  • 50 μL buffer EB was added, tubes were let stand for 1 minute and centrifuged for one minute after
  • flow through kept, eppendorfs labeled pMT1002 plus their colony number

Also see pages 22-23 of the QIAprep Miniprep Handbook for miniprep instructions.

pMT1002 Miniprep Nanodrop values

Plasmid Quantity (ng/μL) 260/280
pMT1002 #1 68.2 1.84
pMT1002 #2 138.7 1.84
pMT1002 #3 127.2 1.88

PCR of miniprepped Barnase

20μL per tube

In each tube:

  • 1μL Forward primer
  • 1μL Reverse primer
  • 0.5μL polymerase
  • 4μL 5X buffer
  • 2μL DNTPs
  • 1μL DNA sample
  • 10.5μL water

3 samples were made for the Barnase

pMT1002 Barnase PCR

Pmt1002barnasePCRattempt1.jpg

B21 Plasmid Digest

put 10x green FD buffer on ice to thaw.

Tube A:

  • 1μL Xba1
  • 1μL Pst1
  • 2μL green 10x FD buffer
  • 3.4μL colony A plasmid (the amount used to get .5μg of plasmid)
  • 12.6μL H2O (to bring the total to 20μL)

Tube B:

  • 1μL Xba1
  • 1μL Pst1
  • 2μL green 10x FD buffer
  • 1.7μL colony B plasmid (the amount used to get .5μg of plasmid)
  • 14.3μL H2O (to bring the total to 20μL)


Pst1 and Xba1 digest of B21 plasmid, lane 4 and 5 cut, 7 and 8 uncut

B21 digest 6-30-10.jpg

Second Attempt


Tube A:

  • 1μL Xba1
  • 1μL Pst1
  • 5μL green 10x FD buffer
  • 12.5μL colony A plasmid (the amount used to get .5μg of plasmid)
  • 30.5μL H2O (to bring the total to 50μL)

Tube B:

  • 1μL Xba1
  • 1μL Pst1
  • 5μL green 10x FD buffer
  • 6.6μL colony B plasmid (the amount used to get .5μg of plasmid)
  • 36.4μL H2O (to bring the total to 50μL)

Ran on a 1% agarose gel, 100 Volts for 30-40 mins.

Lane 2 buffer, lane 8 and 9 Colony B, lanes 15 and 16 colony A

B21plasmiddigest2ndattempt.jpg


Cut out the backbone (upper band, and placed into four eppendorf tubes, labeled 'B21 backbone' and with their respective weight before the gel was added. Placed all four in the iGEM team fence box in the -4°C freezer to be purified tomorrow.

Other

  • Designed primer sequences for ACC synthase degradation site, linker sequence connecting Barnase to the degradation site.
  • Designed primers to amplify germination/embryogenesis promoter and AtArp2 promoter.