Team:Hawaii/Ligation of pRL1383a Parts

From 2008.igem.org

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== Methods ==
== Methods ==
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#<strong> Round 1 of Restriction Digest and Ligation </strong>
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<strong> Restriction Digest </strong>
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:*Each part is digested with both enzymes. For all cases NEBuffer 2 is used.
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:*Reaction Conditions:
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::*5ul Buffer
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::*1ul Enzyme 1 + 1ul Enzyme 2
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::*0.5 ul BSA
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::* Xul water
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::* Yul insert (1ug if possible)
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::* Zul vector (less than 1ug)
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:*Refer to http://openwetware.org/wiki/DNA_ligation for the reasoning behind this.
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:*Running Conditions: 2 hours at 37&deg;C.
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:*Check the progress of the reaction by running a 0.8% gel.
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#<strong>Ligation </strong>
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{| border="1"
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|+ ''' Ligation'''
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!width="100"|Name
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|-
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|rep(80ng)+B0030(25ng)
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|-
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|oriV(20ng)+pSB1A2(50ng)
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|-
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|aadA (BB)
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|}
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#Transformation
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#Verification with Colony PCR
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== Results ==
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===8/11===
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[[Image:re_digest_8_11_08.jpg|right|thumb|300px|Restriction digest from 8/11/08.]]
{| border="1"
{| border="1"
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|n/a
|n/a
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#<strong>Ligation </strong>
 
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{| border="1"
 
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|+ ''' Ligation'''
 
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!width="100"|Name
 
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|-
 
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|rep(80ng)+B0030(25ng)
 
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|-
 
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|oriV(20ng)+pSB1A2(50ng)
 
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|-
 
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|aadA (BB)
 
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|}
 
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#Transformation
 
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#Verification with Colony PCR
 
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== Results ==
 
* <br>
* <br>

Revision as of 02:45, 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
Ligation
Name
rep(80ng)+B0030(25ng)
oriV(20ng)+pSB1A2(50ng)
aadA (BB)
  1. Transformation
  1. Verification with Colony PCR

Results

8/11

Restriction digest from 8/11/08.
Restriction Digest
Name size enzyme quantity date of digest
rep 3.3kb XbaI & PstI 5ng/ul 8/11/08
oriV 415bp XbaI & PstI 5ng/ul 8/11/08
aadA (pRL1383a) 806bp XbaI & PstI 5ng/ul 8/11/08
aadA (BB) 806bp XbaI & PstI 5ng/ul 8/11/08
oriT ~125bp XbaI & PstI n/a 8/27/08
P1 lytic Region 1.3kb EcoRI & SpeI 5ng/ul 8/11/08
[http://partsregistry.org/Part:BBa_B0030 BBa_B0030] 2094bp SpeI & PstI 4ng/ul 8/11/08
[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] 3318bp EcoRI & XbaI 5.7ng/ul 8/11/08
[http://partsregistry.org/Part:pSB1A2 pSB1A2] 2094bp SpeI & PstI 5.5ng/ul 8/11/08
[http://partsregistry.org/Part:BBa_I14032 BBa_I14032] 4462bp n/a n/a

Discussion

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


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