Melbourne/Lab Notebook Weeks 5-8

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*[[Melbourne/Colony PCRl|Colony PCRed]] using [[Melbourne/primary vf2|vf2]] and [[Melbourne/primary vR|vR]] primers the transformations from 20/7. 3 colonies from ligation plate and one from plasmid control plate.
<font size=3><b>24 July 2007  
<font size=3><b>24 July 2007  
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*[[Melbourne/Loading a DNA gel|Ran Gel]] from PCRs (23/7) at 100V.
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-results: colony 3 didn't give product.
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*Streaked out some cells from index plate (No.3).
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*[[Melbourne/Diagnostic Digest|Digested]] minipreprs from 9/7:
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*# [[Melbourne/BBa_E0430|P1 11A]] with X/P
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*# [[Melbourne/BBa_R0082|P1 15P]] with S/P
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(no BSA or AP used).
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*Inactivated enzymes at 80degreesC in a heat block for 10min.
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*[[Melbourne/Ligation Protocol|Ligated]] the two parts together (5ul each) overnight at 4degreesC.
<font size=3><b>31 July 2007  
<font size=3><b>31 July 2007  
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*[[Melbourne/Transformation Protocol|Transformation]] of ligations from 31/7 into NM522 competent cells using 3ul of DMSO.
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*[[Melbourne/Diagnostic Digest|Diagnostic Digest]] of
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*# [[Melbourne/BBa_P1010_AC|P2 21B]]
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*# [[Melbourne/BBa_P1010_AK|P2 23N]]
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*# [[Melbourne/BBa_P1010_A|P3 20I]]
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using miniprep from 13/7. All digests were E/P.
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[[Melbourne/Loading a DNA gel|Ran a Gel]]:
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*# [[Melbourne/primary DNA marker|DNA ladder]]
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*# [[Melbourne/BBa_P1010_AC|P2 21B]]
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*# [[Melbourne/BBa_P1010_AC|P2 21B]]
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*# [[Melbourne/BBa_P1010_AK|P2 23N]]
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*# [[Melbourne/BBa_P1010_AK|P2 23N]]
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*# [[Melbourne/BBa_P1010_A|P3 20I]]
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*# [[Melbourne/BBa_P1010_A|P3 20I]]
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-Results:
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*#
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*# Low digestion efficiency
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*# Low digestion efficiency
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*# No insert seen but DNA present
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*# No insert seen but DNA present
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*# Ok digestion (not very good)
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*# Ok digestion (not very good)
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--Digestion imcomplete??
<font size=3><b>1 Aug 2007  
<font size=3><b>1 Aug 2007  
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*[[Melbourne/Growing up cells|Cultured]] 6 colonies picked from 1/8 transformations. A total of about 500 colonies grew with none on control (competent cells only plate).
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*[[Melbourne/Transformation Protocol|Transformation]] of [[Melbourne/BBa_E0033|P2 9E]] (LacZa with KanR) from iGEM plate.
<font size=3><b>2 Aug 2007  
<font size=3><b>2 Aug 2007  
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*[[Alkaline Lysis Miniprep protocol|Alkaline Lysis Miniprepped]] colonies from 1/8. ([[Melbourne/BBa_E0430|P1 11A]] +  [[Melbourne/BBa_R0082|P1 15P]] ligation. Named OMP-YFP)
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-results: colony 6 had two bands of sizes approximately 1000 and 1100bp.
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*Streaked out colony 6 liquid culture on LBA plate and grew overnight.
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*Picked 5 colonies from streaked out plate from 2/8 and [[Melbourne/Growing up cells|Cultured]] overnight.
<font size=3><b>4 Aug 2007  
<font size=3><b>4 Aug 2007  
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*[[Alkaline Lysis Miniprep protocol|Alkaline Lysis Miniprepped]] colonies from 3/8.
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-All lanes had a single band at 1000-1100bp. Two had a 1000bp band and three a 1100bp band.
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*Colony 3 of above (1100bp band) was [[Melbourne/Miniprep protocol|Miniprepped]] using the Wizerd kit.
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*OD of miniprep measured using a [[Melbourne/DNA concentration measurement|spectrophotometer]] to be 20ng/ul.
==Week 7==
==Week 7==
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*Minipreppred OMP-YFP sent off for sequencing. 10ul of miniprep and 2ul each of [[Melbourne/primary vf2|vf2]] and [[Melbourne/primary vR|vR]] primers (10uM) were sent.
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Sequence was the desired [[Melbourne/BBa_R0082|P1 15P]] to [[Melbourne/BBa_E0430|P1 11A]] ligation product (ampR).
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Part created:
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[[Melbourne/OMP-EYFP|OMP-EYFP]]</font>
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*[[Melbourne/Growing up cells|Liquid cultured]] [[Melbourne/BBa_J61035|P4 8J]] and [[Melbourne/BBa_E0033|P2 9E]] (from 1/8) in Kan.
<font size=3><b>6 Aug 2007  
<font size=3><b>6 Aug 2007  
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*[[Melbourne/Miniprep protocol|Miniprepped]] and *[[Melbourne/Making glycerol Stocksl|created Glycerol Stocks]] of cultures from 5/8.
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*[[Melbourne/Diagnostic Digest|Digested]]:
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*# [[Melbourne/BBa_E0033|P2 9E]] E/P for 1.5h
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*# [[Melbourne/BBa_E0033|P2 9E]] E/X for 2.5h
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*# [[Melbourne/BBa_J61035|P4 8J]] E/S for 2.5h
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*[[Melbourne/Loading a DNA gel|Ran on a Gel]]:
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*# [[Melbourne/primary DNA marker|DNA ladder]]
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*# [[Melbourne/BBa_J61035|P4 8J]] E/S
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*# [[Melbourne/BBa_J61035|P4 8J]] E/S
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*# [[Melbourne/BBa_E0033|P2 9E]] E/P
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*# [[Melbourne/BBa_E0033|P2 9E]] E/P
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*# [[Melbourne/BBa_E0033|P2 9E]] E/X
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*# [[Melbourne/BBa_E0033|P2 9E]] E/X
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*# [[Melbourne/primary DNA marker|DNA ladder]]
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-All above samples seem to be good.
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*Heat inactivated [[Melbourne/BBa_E0033|P2 9E]] E/X and [[Melbourne/BBa_J61035|P4 8J]] E/S at 65degreesC for 10min.
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*[[Melbourne/Ligation Protocol|Ligated]] above DNA fragments together (5ul each) at 4degreesC overnight.
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*[[Melbourne/Transformation Protocol|Transformed]] [[Melbourne/BBa_Q04510|P2 13K]] from iGEM plate =CI. Also transformed ligations from 6/8 (GenR-LacZa) = L.
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*[[Melbourne/Growing up cells|Cultured]] transformants CI 1-4 and L1-3 from above transformations.
<font size=3><b>14 Aug 2007  
<font size=3><b>14 Aug 2007  
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*[[Melbourne/Miniprep protocol|Miniprepped]] CI-2,3,4. CI-1 and L1-3 showed no growth.
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*[[Melbourne/Diagnostic Digest|Digested]] miniprepped DNA for 1.5h:
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*# [[Melbourne/primary DNA marker|DNA ladder]]
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*# CI-2 E/S
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*# CI-2 X/S
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*# CI-3 E/S
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*# CI-3 X/S
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*# CI-4 E/S
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*# CI-4 X/S
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*# [[Melbourne/primary DNA marker|DNA ladder]]
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-CI-2 seems to have the correct DNA.
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*[[Melbourne/Making glycerol Stocksl|Glycerol Stock]] of CI-2 created.
<font size=3><b>15 Aug 2007  
<font size=3><b>15 Aug 2007  
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*[[Melbourne/Diagnostic Digest|Digests]] of CI-2 and a double terminator ([[Melbourne/BBa_B0014|P1 1G]]) were set up.
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*# CI-2 E/S
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*# [[Melbourne/BBa_B0014|P1 1G]] E/X
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and incubated for 2h at 37degreesC.
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*Gel purified 1.8kb band from CI-2 digest and 3kb band from [[Melbourne/BBa_B0014|P1 1G]] digest. These were eluted in 45ul TE buffer.
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*These fragments were then [[Melbourne/Ligation Protocol|Ligated]] together using 5ul of [[Melbourne/BBa_B0014|P1 1G]] vector and 7ul of CI-2 insert, overnight at 4degreesC.
<font size=3><b>18 Aug 2007  
<font size=3><b>18 Aug 2007  
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*[[Melbourne/Transformation Protocol|Transformation]] of above ligation (17/8) onto a Amp + Kan plate. Plate labeled "ligation."

Latest revision as of 07:56, 9 October 2007

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Contents

Week 5

23 July 2007

  • Colony PCRed using vf2 and vR primers the transformations from 20/7. 3 colonies from ligation plate and one from plasmid control plate.

24 July 2007

-results: colony 3 didn't give product.

  • Streaked out some cells from index plate (No.3).


25 July 2007


26 July 2007


27 July 2007

Week 6

30 July 2007

(no BSA or AP used).

  • Inactivated enzymes at 80degreesC in a heat block for 10min.
  • Ligated the two parts together (5ul each) overnight at 4degreesC.

31 July 2007

  • Transformation of ligations from 31/7 into NM522 competent cells using 3ul of DMSO.


using miniprep from 13/7. All digests were E/P.

Ran a Gel:

-Results:

    2. Low digestion efficiency
    3. Low digestion efficiency
    4. No insert seen but DNA present
    5. No insert seen but DNA present
    6. Ok digestion (not very good)
    7. Ok digestion (not very good)

--Digestion imcomplete??

1 Aug 2007

  • Cultured 6 colonies picked from 1/8 transformations. A total of about 500 colonies grew with none on control (competent cells only plate).


2 Aug 2007

-results: colony 6 had two bands of sizes approximately 1000 and 1100bp.

  • Streaked out colony 6 liquid culture on LBA plate and grew overnight.


3 Aug 2007

  • Picked 5 colonies from streaked out plate from 2/8 and Cultured overnight.

4 Aug 2007


-All lanes had a single band at 1000-1100bp. Two had a 1000bp band and three a 1100bp band.

Week 7

5 Aug 2007

  • Minipreppred OMP-YFP sent off for sequencing. 10ul of miniprep and 2ul each of vf2 and vR primers (10uM) were sent.

Sequence was the desired P1 15P to P1 11A ligation product (ampR).


Part created: OMP-EYFP


6 Aug 2007

-All above samples seem to be good.

  • Heat inactivated P2 9E E/X and P4 8J E/S at 65degreesC for 10min.
  • Ligated above DNA fragments together (5ul each) at 4degreesC overnight.


7 Aug 2007


8 Aug 2007


9 Aug 2007


10 Aug 2007


11 Aug 2007

Week 8

12 Aug 2007

  • Transformed P2 13K from iGEM plate =CI. Also transformed ligations from 6/8 (GenR-LacZa) = L.


13 Aug 2007

  • Cultured transformants CI 1-4 and L1-3 from above transformations.

14 Aug 2007

-CI-2 seems to have the correct DNA.

15 Aug 2007


16 Aug 2007


17 Aug 2007

and incubated for 2h at 37degreesC.

  • Gel purified 1.8kb band from CI-2 digest and 3kb band from P1 1G digest. These were eluted in 45ul TE buffer.
  • These fragments were then Ligated together using 5ul of P1 1G vector and 7ul of CI-2 insert, overnight at 4degreesC.

18 Aug 2007

  • Transformation of above ligation (17/8) onto a Amp + Kan plate. Plate labeled "ligation."
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