Melbourne/Lab Notebook Weeks 5-8

From 2007.igem.org

(Difference between revisions)
(Week 7)
(Week 8)
Line 184: Line 184:
</b>
</b>
</font><BR>
</font><BR>
 +
 +
*[[Melbourne/Transformation Protocol|Transformed]] [[Melbourne/BBa_Q04510|P2 13K]] from iGEM plate =CI. Also transformed ligations from 6/8 (GenR-LacZa) = L.
Line 190: Line 192:
</font><BR>
</font><BR>
 +
*[[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  
Line 195: Line 198:
</font><BR>
</font><BR>
 +
*[[Melbourne/Miniprep protocol|Miniprepped]] CI-2,3,4. CI-1 and L1-3 showed no growth.
 +
*[[Melbourne/Diagnostic Digest|Digested]] miniprepped DNA for 1.5h:
 +
*# [[Melbourne/primary DNA marker|DNA ladder]]
 +
*# CI-2 E/S
 +
*# CI-2 X/S
 +
*# CI-3 E/S
 +
*# CI-3 X/S
 +
*# CI-4 E/S
 +
*# CI-4 X/S
 +
*# [[Melbourne/primary DNA marker|DNA ladder]]
 +
 +
-CI-2 seems to have the correct DNA.
 +
*[[Melbourne/Making glycerol Stocksl|Glycerol Stock]] of CI-2 created.
<font size=3><b>15 Aug 2007  
<font size=3><b>15 Aug 2007  

Revision as of 13:57, 8 October 2007

<Return to Lab notebook> <team home page>

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:

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


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


18 Aug 2007