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July 1st, 2009

  • Started overnight culture of BB1 in 25 ml of LB

July 2nd, 2009

  • Made fresh litre of LB
  • started log growth of BB1
    • 1ml of overnight in 100 ml of Autoclaved LB with 100ul of stock amp solution
  • incubating at 37C
  • started overnight culture of BB2, 3, 4, 5, and pSB1AT3 in 25 ml of LB per colony / plate
  • miniprepped BB1 -=> 64.8ng/ul. Stored in the -20 fridge
  • made 2 tubes of BB1 stock w/ glycerol, stored in -80 freezer

July 3, 2009

  1. Started log phase growth for BB2, 3, 4, 5 in 100 ml of LB with Amp (100 ul)
    • BB2, 3, 4 appeared to be contaminated as there was no significant increase in absorbency. We suspect the source of contamination was from the pipette tip when adding the ampicillin to the LB. The tip had been used repeatedly.
    • Due to the contamination observed, the ampicillin may also have been contaminated. If the same situation occurs again, it may be due to this.
  2. Miniprepped BB5 ([] = 3.54.4 ng/ul) and froze 2 cryotubes for long term stock

July 5, 2009

  1. C plasmid mislabelled
  2. Overnight of 2, 3, 4 tet started
  3. 1, 5, C, Kan were digested - 250 ng of DNA
    • overnight @ 37 degrees celcius

July 6, 2009

  1. 2, 3, 4 overnight cultures did not grow well - replated 2, 3, 4 on new Amp plates
  2. Gel image (~1hr @ 120 V)
    • L1: Ladder - too concentrated (not well resolved) probably because gel was made with 0.5X TBE while loading buffer for gel was 1X TBE - next time use 0.5X
    • L2: BB1 - not resolved, appears to not be fully digested, too much DNA? Appears to be proper plasmid, but a part ran off. Next time run gel for less time (~30 min)
    • L3: BB5 - nothing appeared
    • L4: C - looks very faint, add more next time (30 ul instead of 20 ul)
    • K - same as C
  3. Redigested BB5 - followed digestion protocol suggested in BioBrick Assembly Manual
    • Digestion mixture: 500 ng plasmid ([BB5] = 328.0 ng/ul, used 1.5 ul), 1 ul EcoRI-HF, Spel each, 10X NE Buffer 2 5 ul, 100X BSA 0.5 ul, made up to 50 ul
  4. After nearly 4 hours in the incubator, the tet plasmid culture had an absorbency of 0.011. We decided to leave it overnight and to miniprep tomorrow.

July 7, 2009

  1. Absorbency of tet plasmid this morning = 0.62
    • Made 2 cryotubes of stock (put in -80 freezer), miniprepped [] = 56.4 ng/ul
  2. After 30 minutes of running the gel at 97 mV, the bands could be seen but there was not enough separation. We continued to run it.
  3. Started overnight culture for BB2, 3, 4 in 100 ul, 130 ul, and 100 ul of LB
  4. After 1h 45min, gel was examined:
    • Comparing lanes 2 and 3 (BB1 digested vs. undigested), we conclude that BB1 is the correct size and results of digested are as expected
    • Lanes 4, 5 had very faint bands. Since they were digests of BB5 done on Sunday and repeated Monday, we conclude the digestion time may be too long such that most DNA had been degraded
    • Lane 6 was undigested BB5; the band shows the DNA sample was not degraded
    • Lane 7 and 8 were reruns of C and K plasmids. Although bands were faint, they were the size expected
    • Future adjustments:
    • 2% agarose was too thick because after running for nearly 2 hr, the separation did not happen. Will try 1.5% agarose next time
    • Digestion time for BB5 should be shortened

July 8, 2009

  1. Started log phase growth for BB2, 3, 4
  2. Transfected DB3.1 cells with BB6 (R0040) and the operation vector (pSB3C5) and plated them on appropriate antibiotic plates
  3. Miniprepped BB2, 3, 4 from overnight culture
  4. BB2, 3, 4 stocked in cryotubes with glycerol from log phase culture
  5. Poured 8 Amp plates

July 9, 2009

  1. Digested BB2, 3, 4, tet, and 5 for 1h and 3h
  2. Plated BB1, 5, C, K from stock tubes
  3. Transfected cell plates from yesterday did not show any colonies. We therefore plated the remaining liquid culture (stored at 4 degrees Celsius) onto appropriate antibiotic plkates
  4. Ran a gel of the digests
    • Loaded 20 ul of sample in each well
    • Used all of 50 ml agarose + TBE to make gel to make deep wells
    • 1h digests were kept on ice while waiting for 3h digests
    • DNA ladder was heat shocked at 60 degrees Celsius for 3 min before mixing with dye and TBE
  • Gel turned out well though there are still problems with the ladder - suggest loading small amount of DNA ladder in a small well
  • There is a minimal difference between 1h and 3h digests - suggest we stick with 1h digest in the same conditions. Also, future - if there is a free well, try 30 min digest

July 10, 2009

  1. All 4 replates from yesterday grew colonies (1, 5, C, K), indicating our stock tubes do indeed contain the plasmids
  2. Replated BB2, 3, 4, Tet from stock tubes
  3. Digested BB1, 2, 5 and ran on 1.3% TAE gel with 100 bp DNA ladder
    • 10 min and 1h digests don't show a big difference and we should try a 10 min digest in future
    • BB1 appears to have contaminations as the top band was not at expected location
    • BB5 did not appear on the gel again. We suspect the plasmids are contaminated with DNase or other proteins. We will run another gel with undigested BB5 after incubating @ 37 C on Monday
    • The small fragments of the biobricks were not seen on the gel. Austin said this was expected.

July 13, 2009

  1. Replates of BB2, 3, 4, Tet are growing well
  2. Started overnight culture of BB6 and 0 with antibiotics
  3. Transfected DH5-a cells with BB7, BB1, 5 and TM0785 and plated on Amp plates (2 plates for each BB)

July 14, 2009

  • Ran a gel for PCR products & undigested BB5
  • started overnight for TM0785, BB1, 6,5, and replated BB7

July 15, 2009

  • started overnight for 5
  • started log phase growth for 5
  • miniprepped log phase culture
  • stocked 2 cryotubes of 1
  • made a mix of 100 bp ladder and buffer
  • Ran a PCR reaction for sample showed non-specific band and run on a gel
  • Digested Mini-prepped TM0285 and ran on gel
    • pour digest will redo tomorrow

July 16, 2009

  • ran 1st PCR according to protocol, increased MgCl2 by 0.5mmol
  • ran a TD-PCR reaction
  • Ran a 2nd PCR, with more primer (used 0.5)
  • Ran another TD PCR in same conditions as befoe
  • Ran a negative control for PCR, which included everything except template.

July 17, 2009

  • Ran a gel for PCR P5 + N6
  • ran a gel for BB1 BB5 and TM0785
  • miniprepped BB7 from log phase
    • Made stock of BB7 as well

July 20, 2009

  • Overnight PCR in PCR machine --> gDNA --> TD
  • Completed Day 1 assemblies for the control module 1+2, 3+2, and 4+5
  • 2+3 and 4+5 ligations, we change the
  • we also transfected DN5 cells with all of the ligate instead of 2ul suggested by the NEB protocol

July 21, 2009

  • PCR of gDNA was successful 800bp band was observed on the 1.3% TAE agarose gel with the 100 bp ladder
  • A second gel was run with the rest of the product
    • however the well loaded into was broken and some of the sample was lost/ spread into 2 adjacent wells (see pic)
    • and excised sample was taken and purified. It was nanodropped at 11.1ng/ul
  • BB7 was digested an run in lane 3 however no bands were visible
  • K and C plates were poured and are drying in the incubator
  • assembly plates were checked, however no growth was visible (there were weird bumps on the plate that maybe colonies but they looked weird will review tomorrow)

July 22, 2009

  • digested + ligated encapsulin into a C backbone and transformed DH5 stocks
  • re-plated left over 1+2 and 3+2 and 4+5

July 23, 2009

  • observed july 22 plates --> no growth
  • suggest redoing digest
  • digesting Encapsulin
    • run gel to observe
  • Due to lack of band in the C plasmid digest a libation was not preformed. The encapsulin digest will be stored in a 4C fridge for the next day
  • Lack of a band in both C plasmid lanes
    • Contamination
    • Lack of DNA
    • small volume loaded = no DNA?

July 24, 2009

  • Digested C and K plasmids with EcoRI and PstI in the PCR machine
  • Also prepared negative controls
  • Ran Digest and controls on gel / 100bp lader and HindIII digest
    • C plasmid could be observed with a expected 3k band
    • K plasmid digest did not yield a band
  • Started overnight for encapsulin transfected cells

July 27, 2009

  • Digested encapsulin plasmid and BB7 plasmid with -ve controls and straight from fridge control
  • imaged gel showed nothing in the digest lane and a weak signal from the bands in the control lanes
  • Also through discussion with calvin it was discovered that our mini preps are producing really low yields of plasmid
  • Also through discussion with Calving we have determined that we should do future ligations in smaller volumes, Ligation reactions do NOT scale up well as our plates showed no colonies.
  • Started overnights of BB1, 2, 3, 4, 5, 6, 7, Tet, C

July 28, 2009

  • miniprepped BB1, 5, 7, C and Tet but yield was very low ~20ng/ul
  • Calvin also miniprepped one sample for us to see if our miniprep kit was the problem. His miniprep idled 45 ng/ml
  • One problem with our elute was greater than the volume of TE we put in for elution. Either there was still wash buffer in the spin column or there was something wrong with the pipetting.
  • We redid the mini prep with 50 ul of TE for elution for BB7, Yield was 16.3 ng/ul. Since the yield was so low, we decided to regrow an overnight culture in 5ml of LB and miniprep those tomorrow.

July 29, 2009

  • Ran gel with PCR encapsulin and gel extract [] of 11.0 ng/ul was obtained
  • Ran gel with K plasmid from storage found a sufficient [] however there was no ladder and the length is questionable.
  • Ran minipreps on 1, 3, 4, 5, Tet and C.
    • changed protocol to use 5mL of overnight culture
    • great yield eluted w/ 50 ul of TE
  • digested BB 4+5 and doing overnight ligations in the PCR machine at 16C
  • ran gel of 4+5 digests

July 30, 2009

  • at the end of the day a gel was run and all bands were consistent and a great improvement to our initial gels i.e. May/June. Ladders did not show due to Kenny forgetting to add the ladder to the stock.
  • earlier in the day we took the ligation of 4+5 and encapsulin and transformed the DH5 with them. The encapsulin transformants were done in a pBSK plasmid and plated on a gal + IPTG + LB + amp plate. 30 ul of IPTG and 50 ul of gal and 50ul of LB were mixed and spread on an Amp plate. The transformants were then plated onto.
  • 4+5 transformants were plated on OLD (Jul. 3) tet plates.
  • A ligation of 1+2 and 3+2 was performed and is in Rommens PCR machine 3 for tomorrow.

July 31, 2009

  • Transfected 2ul of 1+2 and 3+2 ligation prduct into 25 ul of competent cells each. Plated on Amp plates
  • ran a gel with the ligation products (10ul)