Team:Hawaii/Ligation of pRL1383a Parts

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|rep(80ng)+B0030(25ng)
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|oriV(20ng)+pSB1A2(50ng)
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|aadA (BB)
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#Transformation
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|rep(20ng)+B0030(16ng)
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|0.253:1
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|oriV(20ng)+pSB1A2(50ng)
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== Discussion ==
== Discussion ==

Revision as of 03:03, 14 August 2008

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Contents

Ligation of Parts

  • The BioBrick parts of pRL1383a are to be ligated in a series of experiments.


Methods

Restriction Digest

  • Each part is digested with both enzymes. For all cases NEBuffer 2 is used.
  • Reaction Conditions:
  • 5ul Buffer
  • 1ul Enzyme 1 + 1ul Enzyme 2
  • 0.5 ul BSA
  • Xul water
  • Yul insert (1ug if possible)
  • Zul vector (less than 1ug)
  • Refer to http://openwetware.org/wiki/DNA_ligation for the reasoning behind this.
  • Running Conditions: 2 hours at 37°C.
  • Check the progress of the reaction by running a 0.8% gel.
  1. Ligation


  1. Transformation
  1. Verification with Colony PCR

Results

7/27

  • A few re-digests and ligations were performed this day: oriT + pSB1A3, oriV + pSB1A3, mob + B0024, rep + R0010, aadA(pRL1383a+R0010). Afterwards, the ligation product was transformed to DH5-alpha. Only OriT transformed. I did not check the progress of this experiment with a gel, so I am not sure where this went wrong.
  • Next time, a gel will be run after every step.
Restriction Digest
Name size enzyme quantity
oriT ~125bp XbaI & PstI n/a

8/11

Restriction digest from 8/11/08.
Restriction Digest
Name size enzyme quantity
rep 3.3kb XbaI & PstI 5ng/ul
oriV 415bp XbaI & PstI 5ng/ul
aadA (pRL1383a) 806bp XbaI & PstI 5ng/ul
aadA (BB) 806bp XbaI & PstI 5ng/ul
P1 lytic Region 1.3kb EcoRI & SpeI 5ng/ul
[http://partsregistry.org/Part:BBa_B0030 BBa_B0030] 2094bp SpeI & PstI 4ng/ul
[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] 3318bp EcoRI & XbaI 5.7ng/ul
[http://partsregistry.org/Part:pSB1A2 pSB1A2] 2094bp SpeI & PstI 5.5ng/ul
Ligation
Name
rep(20ng)+B0030(16ng) 0.253:1
oriV(20ng)+pSB1A2(50ng)
aadA (BB)

Discussion

  • What was learned and how to do future experiments differently.


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