Team:Hawaii/Construction of BioBrick intermediates

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(Difference between revisions)
(New page: {{Team:Hawaii/Header}} == Construction of BioBrick intermediates == * Construct: ::* ''nir'' + rbs ::* GFP + tt ::* GFPf + tt == Methods == ===Attempt #1=== ====Restriction digest==== :*...)
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== Results ==
== Results ==
===Attempt #1===
===Attempt #1===
 +
====Transformation====
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<div style="text-align: center;"> '''Colony forming units observed'''</div>
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{| border="1" class="wikitable"
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! Construct
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! CFUs
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|-
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| align="center"|''nir''+B0034
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| align="center"|0<sup>1</sup>
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|-
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| align="center"|GFP + B0024
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| align="center"|5
 +
|-
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| align="center"|GFPf + B0024
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| align="center"|15
 +
|-
 +
|}
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<sup>1</sup> Though no colonies were initially observed, when the plate was scraped and restreaked (to purify), colonies were observed on the new LB+amp plate.
 +
====Restriction digest of plasmid preps (to verify insert)====
 +
[[Image:plasmidpreps072508.jpeg|right|thumb|200px|EtBr stained 1.2% agarose gel. Twenty microliters of each restriction digest reaction were loaded into each well.]]
 +
The colony PCR was loaded onto a 4% gel so bands did not resolve. Since a plasmid prep was already done for the parts, the plasmid preps were RE digested with EcoRI to verify insertion of GFP(f) (Did not have room to load nir). The bands, expected to be ~2900bp, were both ~2100bp, indicating the GFP was not inserted. We likely digested with the wrong enzymes (XbaI and SpeI instead of EcoRI and XbaI) so the vector self ligated without the GFP(f) or tt present.
== Discussion ==
== Discussion ==

Revision as of 21:15, 29 July 2008

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Contents

Construction of BioBrick intermediates

  • Construct:
  • nir + rbs
  • GFP + tt
  • GFPf + tt

Methods

Attempt #1

Restriction digest

  • Digested nir, GFP, and GFPf with EcoRI and SpeI in NEBuffer 2
  • Digested B0034 (rbs) and B0024 (tt) with EcoRI and XbaI in NEBuffer 2 with BSA

Gel purification

  • Ran digest reaction on an EtBr stained 2% gel to extract bands of interest

Ligated parts

  • Ligated parts in 20 mu;l reactions:
  • 4 μl B0034 (8.8 ng) + 2.42 μl nir (26.378 ng)
  • 1 μl B0024 (15.5. ng) + 8 μl GFP (41.6 ng)
  • 0.4 μl B0024 (6.2 ng) + 1.8 μl GFPf (1.8 ng)

Subcloned in DH5α

  • Used 5 μl ligation reaction to transform
  • Incubated at 37C with shaking for 100 min.

Colony PCR verification of constructs

Attempt #2

Restriction digest

  • Digested nir, GFP, and GFPf with EcoRI and SpeI in NEBuffer EcoRI with BSA
  • Sequentially digested B0034 (rbs) and B0024 (tt) with XbaI and EcoRI in their respective buffers

Gel purification

  • Ran digest reaction on an EtBr stained 1.2% gel to extract bands of interest

Ligated parts

  • Ligated parts in 20 mu;l reactions:

Subcloned in DB3.1

  • Used 5 μl ligation reaction to transform

Results

Attempt #1

Transformation

Colony forming units observed
Construct CFUs
nir+B0034 01
GFP + B0024 5
GFPf + B0024 15

1 Though no colonies were initially observed, when the plate was scraped and restreaked (to purify), colonies were observed on the new LB+amp plate.

Restriction digest of plasmid preps (to verify insert)

EtBr stained 1.2% agarose gel. Twenty microliters of each restriction digest reaction were loaded into each well.

The colony PCR was loaded onto a 4% gel so bands did not resolve. Since a plasmid prep was already done for the parts, the plasmid preps were RE digested with EcoRI to verify insertion of GFP(f) (Did not have room to load nir). The bands, expected to be ~2900bp, were both ~2100bp, indicating the GFP was not inserted. We likely digested with the wrong enzymes (XbaI and SpeI instead of EcoRI and XbaI) so the vector self ligated without the GFP(f) or tt present.

Discussion

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


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