Difference between revisions of "Part:BBa I751311:Design"

 
(→‎Design Notes)
 
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===Design Notes===
 
===Design Notes===
 +
*'''Eco/Nhe assembly'''
 +
1. Cut Biobrick part by EcoRI and SpeI.
  
−
+
2. Cut pBR322 by EcoRI and NheI.
  
 +
3. Ligation and TF.
  
 +
pBR322 has NheI site downstream of EcoRI site.
 +
 +
NheI sequence is "'''G^CTAGC'''", and SpeI sequence is "'''A^CTAGT'''". They have the sequence "'''CTAG'''" in common. So, NheI fragment and SpeI fragment can be ligated. However, this procedure kills SpeI site, and you can't assemble parts anymore. (EcoRI and XbaI site is still alive, so you can assemble upward)
 +
 +
This procedure allowed us to introduce any Biobrick part into pBR322 plasmid.
 +
 +
 +
'''*ATTENTION'''
 +
We made this part  by introducing pcI oligo nucleotide into BBa_I751310.
 +
 +
In both ends, EcoRI fragment is added to this pcI oligo. So, this part sequence is different from shown in this page.
 +
 +
This part sequence is actually like the following:
 +
 +
Eco-pcI-Eco-Xba-rbs-GFP-terminator
  
 
===Source===
 
===Source===

Latest revision as of 04:32, 27 October 2007


pBR322 pcI-GFP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 750


Design Notes

  • Eco/Nhe assembly

1. Cut Biobrick part by EcoRI and SpeI.

2. Cut pBR322 by EcoRI and NheI.

3. Ligation and TF.

pBR322 has NheI site downstream of EcoRI site.

NheI sequence is "G^CTAGC", and SpeI sequence is "A^CTAGT". They have the sequence "CTAG" in common. So, NheI fragment and SpeI fragment can be ligated. However, this procedure kills SpeI site, and you can't assemble parts anymore. (EcoRI and XbaI site is still alive, so you can assemble upward)

This procedure allowed us to introduce any Biobrick part into pBR322 plasmid.


*ATTENTION We made this part by introducing pcI oligo nucleotide into BBa_I751310.

In both ends, EcoRI fragment is added to this pcI oligo. So, this part sequence is different from shown in this page.

This part sequence is actually like the following:

Eco-pcI-Eco-Xba-rbs-GFP-terminator

Source

References