From 2009.igem.org

Latest revision as of 18:16, 21 October 2009

June 3, 2009

1. Plasmid transformed = pSB1AC3 (TEST)
   • Note: team members were of mixed opinion as to whether the transform was likely due to low availability of DNA - DNA was split between a number of transformation reactions for at least 2 different DB3.1 strains. Plates incubated at 30 degrees Celsius overnight, temp. raised to 37 degrees approx. 10:30 a.m.

June 5, 2009

1. Tet plates made

   Recipe for 200 ml (approx. 10 plates):

   2.2 g agar in 200 ml fresh LB

   Note: do not re-autoclave LB, it will caramelize!

   Recipe for 200 ml LB:

   a) 1 g yeast extract

   b) 2 g peptotryptone

   c) 2 g NaCl

   d) 200 ml water

2. Autoclaved at 11:20 a.m., picked up at 1:15 p.m. and cooled in water bath at 37 degrees Celsius
3. Added 200 microlitres tetracycline stock (from -20 freezer) to final concentration of 20 microgram/ml
4. Swirl and poured into prepared, labeled plates
   • Note: label on plates reads June 3, 2009 but should be June 5, 2009 (8 plates in total - either I overpoured or these plates are a bit larger than I was used to)
5. Inverted and put in 37 degree incubator to dry

June 8, 2009

1. Bacteria grown to log phase using sterile inoculating loop to inoculate approx. 200 ml of LB media with bacteria from 2 plates (approx. 3 colonies)
2. Bacterial liquid culture placed in shaker at 10:51 a.m.

June 9, 2009

1. Digested miniprepped gel with EcoRI and SpeI
2. Running gel to see if correct plasmid was purified - should expect to see bands at ~3200 bp
3. DNA Ladder made - 6 microlitres of stock used

June 10, 2009

1. Poured 10 Tet plates following procedure on June 5, 2009
2. Due to problems with DNA ladder and restriction enzyme digest yesterday, procedures were followed again today with the following adjustments
   • DNA was diluted and run on lanes 1-5 of gel:
     • Lane 1 - 1X
     • Lane 2 - 1/6X
     • Lane 3 - 1/36X
     • Lane 4 - 1/10X
     • Lane 5 - 1/100X
   • Restriction enzyme digest was repeated with an incubation time of 4 h at 37 degrees Celsius and heat shock at 80 degrees Celsius was done in hot water bath
   • Results: Dilution did not work well (ladder could not be visualized). Digest appeared to work as 3 bands could be seen in last 2 lanes
   • Adjustments for tomorrow:
     • Spin down enzymes before using
     • Overnight digest

June 11, 2009

• We did not have enough isolated plasmid DNA to justify doing another gel so we decided to perform another gel so we decided to perform another miniprep to get some more
• Used DB3.1 transformed bacteria from shaker(measured spec 0.794)
• Miniprep
  • potential issue our microcentrifuge only spins @ 10,000 not required 12,000
• Binding DNA
  • did both optional steps preheated TE + washed w/ W10

• measured UV absorbance = 4.2ng/ul
• in all there is ~ 74 ul of TE should indicated ~370 ng of plasmid.
• Digest
  • NOTE: plasmid length w/e biobric is 3189 bp biobrick length is 3255bp this can explain lack of 2 clear bands on gel after ligation.
  • nevertheless, we will perform another Gel to make sure we can properly run ladder to determine fragment sizes

• digest measurements: 250ng plasmid all else half except BSA

June 12, 2009

• Ran Gel
  • Ladder and other bands were able to be visualized however were still a little wonky
  • Suggests for improvement
    • increase the glycerol [] in loading dye as there have been problems with diffusion when loading samples as the solution is not falling into the wells

June 15, 2009

• made 1ml and 200ul aliquotes of Kanamycin 10mg/ml stock and stored at -20C
  • Recipe: 250ug Kanamycin + 25ml sterile water
  • USE : 2x200ul for final working concentration of 20ug/ml

Trouble shooting ladder

• it was noted that the 1x TBE buffer recipe was off. The recipe that we were using called for too much Boric acid. After reviewing the literature a new recipe which contained:
  • 54g of Tris base
  • 27.5g of Boric Acid
  • 20ml of 0.5 M EDTA (ph 8.0)
  • 800ml ddH2O

• It is also recommended that we discard our 1x TBE and 10x TBE stocks.

=June 16, 2009+

• poured 8 K plates and 8 C plates. Followed protocol of "pouring agar plates with antibiotics"
• Antibiotic amounts ere calculated:
  • Kanamycin
    • working concentration = 20ug/ul
    • amount required = 20 ug/ml X 200ul = 4000 ug = 4mg
    • stock [] = 10 mg/ml
    • volume of stock required = (4mg/10mg)*1ml = 0.4ml = 400ul
    • 400ul/200ul = 2 aliquots of 200 ul required

• • Chloramphnicol
    • working concentration = 25ug/ul
    • amount required = 25 ug/ml X 200ul = 5000 ug = 4mg
    • stock [] = 25 mg/ml
    • volume of stock required = (5mg/25mg)*1ml = 0.2ml = 200ul
    • 200ul/200ul = 1 aliquot of 200 ul required

June 17,2009

• Due to mix up of # of aliquots yesterday, we poured 8 more plates of K and 8 more plates of C this time at the correct concentrations.
• received the new DNA ladder, and we ran it on a gel to compare with the old ladder.

June 18, 2009

• Transformed first 2 plasmid backbones in the biobrick assembly procedure pSB1AC3 and pSB1AT3
• Note;
  • We used sorval tubes when adding LV and Glucose and incubating on the shaker
  • We flamed the spreader to sterilize it before spreading the cells on the agar + antibiotic plate.

• Made up 50 ml of LV + 20mM glucose for future use and will place in fridge tomorrow if there is no bacterial growth after storing at room temperature for a day.
• will grow cells up to log phase tomorrow for miniprep and freezing
• left over cells from incubation on shaker were stored in the 4 degree fridge over night

June 21, 2009

• Inoculated 1 colony from each plate of transformed DB3.1 cells into 50 ml of LB
• Placed the flasks in the shaker to grow an overnight culture
• THere were no obvious colonies on the Tet. plate. Instead the cells seemed to be spread out over a large area, this maybe due to the low temperature when the plates were poured=== the agar was already polymerizing resulting in an uneven surface.
• A small area of cells in the Tet plates were inoculated.

June 22, 2009

• inoculated the overnight culture into antibiotic-containing LB medium and grew to log phase
• Cells incubated at 40 celcius grew considerably quicker than cells incubated at 37 celcius:
• After a growing time of 5.5 hours the absorbance of Tet and Camp were doubled.
• the Tet cells did not grow well in antibiotics ths they were left in the incubator and not frozen or refrigerated.
• C cells were frozen down in 2 cryotubes in a final glycerol concentration of 20%

June 23, 2009

• DB 3.1 was transformed with backbone plasmid pSB1AK3 and plated on premade K plates
• Tet plates were remade as well
• K and Tet plates were placed in the incubator
• Mini-preped 40 C Chloroamphenicol observed a result of 72.7ng/ul in a total of 75 ul
• plates were placed in the incubator

June 24, 2009

• Amp plates were poured 500 ul of Amp stock / 500 ul
• K plates doing well colonies observed
• Tet plates not so good spotted maybe 1 colony
• transformed DB3.1 with BB1 ad BB2
• plated them on amp plates.

June 25, 2009

• Oberseved plates in incubator
  • K plates: ready for overnight
  • et plates: shows 1 or 2 colonies
  • Amp 1 + Amp 2 plate: Very few colonies
    • Transformation efficiency of flash frozen EtOH cells seems to be low (rom obs only)

June 26, 2009

• Transformed BB5 to DB 3.1 Cells and plated on 2 K plates
• Overnight cutter of pSB1AK3 plasmid was inoculated to a liquid LB medium and grown to log phase for Mini-prep and freezing down.
• Miniprepped pSB1AK3 stored in -20 fridge
• stocked cells stored in the -80 freezer

June 29, 2009

• All plates were looked at and were all growing okay
• all plates were moved to the lab bench to sow down bacterial growth
• An overnight growth of all the et resistant plasmid was started
• this needs to be minipreped and frozen down after log growth
• The rest of the plates should be left on the bench till thursday
• miniprep kits labelled with simple / basic instructions
• full instructions were left in the protocol binder.
• log growth requires antibiotics at a same concentration as the agar
• If no growth in log phase culture there is a replete in the incubator on the mid-shelf

June 30, 2009

• removed overnights of et resistant plasmids from the incubator
  • placed autoclaved LB from 4 degree fridge at 37C water bath to warm for 30 min
  • inoculated 2x100 ml LB each with 2ml of overnight culture
  • incubate with shaking at 40C
  • if they grow then stock cells in the freezer
• plasmid mini-prep
  • decided to do minipres of both tubes in case either of the corresponding flasks don't grow
  • per protocol on invitrogen kit
  • placed at 4c for 1.5 - 2.0 hours
  • Attempt to quantify using nanodrop reader.
  • Sample 1 contained 16.4 ng/ul
  • Sample 2 contained 18.4 ng/ul
• Restriction Enzyme Digest
  • to use 25 ul of plasmid miniprep
• we think it is bizarre that a plasmid that is supposed to be et resistant up to 150 ug/ml will not grow in culture at 10ug/ml of et. Since we are selecting this plasmid particularly for its et resistance for use in assembly we feel it would be best to discard this culture and pick another well from the item library perhaps 8A or 15 A. Decided not to proceed with enzyme digest.

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