{{ construct.name }}

http://django.gibthon.org/gibthon/{{ construct.id }}/{{ construct.name }}/
{{ construct.description|linebreaks }}

Fragments: {{ construct.fragments.all.count }}

    {% for f in construct.fragments.all %}
  1. {{ f.name }} {{ f.description }}
  2. {% endfor %}

Features: {{construct.feature_count }}

    {% for f in construct.features_pretty %}
  1. {{ f }}
  2. {% endfor %}

Total lenght: {{ construct.length }} bp

Generated by Gibthon Construct Designer


Files included in this archive

• {{ construct.name }}.pdf - this file
• {{ construct.name }}.gb - genbank file of construct
• {{ construct.name }}-primers.csv - csv file of primers
• PCR/ - openPCR config files
{% for f in construct.fragments.all %}    » {{ construct.name }}-{{ f.name }}.pcr
{% endfor %}

Gibson Assembly Protocol:{{ construct.name }}


Protocol

  1. Extend each fragment using the PCR protocols below
  2. Make up the Assembly Mix with the PCR products
  3. Incubate the Assembly Mix for 1 hour at 50°C
  4. Purify/transform

{% for f in construct.fragments.all %} {% endfor %}

Assembly Mix

Volume
Gibson Master Mix15 μl
{{ f.name }} (extended){{ each|floatformat:1 }} μl
Total20 μl
 

Gibson Master Mix

Volume
Taq ligase (40u/µl)50 μl
5x isothermal buffer100 μl
T5 exonuclease (1u/µl)2 μl
Phusion polymerase (2u/µl)6.25 μl
Nuclease-free water216.75 μl
Total375 μl
 

5x isothermal buffer

Volume
25% PEG-80000.75g
500 mM Tris-HCl pH 7.51500 μl
50mM MgCl275 μl
50mM DTT150 μl
1mM dATP30 μl
1mM dTTP30 μl
1mM dCTP30 μl
1mM dGTP30 μl
5mM NAD300 μl
Nuclease-free water...
Total3000 μl
{% for cf in construct.cf.all %}

{{ cf.fragment.name }}:{{ construct.name }}

Primer datasheet

Primers

{% with primer=cf.primer_top %}

{{ primer.name }}

SectionFullAnneal
Length{{ primer.length }} bp{{ primer.stick.length }} bp
Tm{{ primer.tm|floatformat }}°C{{ primer.stick.tm|floatformat }}°C
Sequence{{ primer.seq_pretty }}
{% endwith %}
{% with primer=cf.primer_bottom %}

{{ primer.name }}

SectionFullAnneal
Length{{ primer.length }} bp{{ primer.stick.length }} bp
Tm{{ primer.tm|floatformat }}°C{{ primer.stick.tm|floatformat }}°C
Sequence{{ primer.seq_pretty }}
{% endwith %}

PCR Protocol

PCR Mix

Volume
Buffer{{ construct.pcrsettings.buffer_v|floatformat:1 }} μl
dNTPs{{ construct.pcrsettings.dntp_v|floatformat:1 }} μl
{{ cf.primer_top.name }}{{ cf.primer_top.vol|floatformat:1 }} μl
{{ cf.primer_bottom.name }}{{ cf.primer_bottom.vol|floatformat:1 }} μl
{{ cf.fragment.name }}{{ cf.vol|floatformat:1 }} μl
Enzyme{{ construct.pcrsettings.enzyme_v|floatformat:1 }} μl
Water{{ cf.water_v|floatformat:1 }} μl
Total Volume{{ construct.pcrsettings.total_v|floatformat:1 }} μl


PCR Instructions

TemperatureTime
Initial Melting98°C30s
cycle 30 times
Melting98°C30s
Annealing{{ cf.tm|floatformat:0 }}°C30s
Elongation72°C{{ cf.time|floatformat:0 }}s
end cycle
Final Elongation98°C7m 30s
Final Hold98°C
{% endfor %}
Created by {{ user.username }} on {% now "jS F Y H:i" %}