Difference between revisions of "HPP New Start and Finish"

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(Created page with 'July 23, 2011 New Oligos to Order START_TOP (67 nt) AATTCGGTCTCAGACGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGTGGAGAGACGCTGCA START_Bot (59 nt) GCGTCTCTCCACGCTAGCACTGTACCTAGGACTGAGC…')
 
 
(21 intermediate revisions by 2 users not shown)
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July 23, 2011
+
'''New START Half Edge'''
−
New Oligos to Order
+
# Use BsaI to free up new START word (GTCC) on 5’ end that allows ligation to new HPP vector.
−
START_TOP (67 nt)
+
# Use BsmBI to free up half-edge word (GTGG) on 3’ end that allows ligation to any Gene1 half-edge.
 +
# In between these is a J23100 promoter.
 +
# Flanked by EcoRI and PstI sticky ends for cloning.<br><br>
  
−
AATTCGGTCTCAGACGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGTGGAGAGACGCTGCA
+
  RI  BsaI>  START              J23100              Half  <BsmBI  PstI<br>
−
 
+
AATTC GGTCTC A GTCC TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC GTGG A GAGACG CTGCA<br>
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START_Bot (59 nt)
+
    G CCAGAG T CAGG AACTGCCGATCGAGTCAGGATCCATGTCACGATCG CACC T CTCTGC G
−
 
+
               
−
GCGTCTCTCCACGCTAGCACTGTACCTAGGACTGAGCTAGCCGTCAACGTCTGAGACCG
+
<br><br>
−
 
+
−
FINISH_Top (40 nt)
+
'''New FINISH Half Edge'''
 +
# Use BsmBI to free up half-edge word (GTGG) on 5’ end that allows ligation to any Gene2 half-edge.
 +
# Use BsaI to free up new FINISH word (CGAG) on 3’ end that allows ligation to new tr5f4HPP vector.
 +
# In between these is a 8 nt spacer.
 +
# Flanked by EcoRI and PstI sticky ends for cloning.<br><br>
  
−
AATTCCGTCTCAGTGGGCATCAGTCGAGAGAGACCCTGCA
+
  RI  BsmBI>    Half  Spacer  FINISH  <BsaI  PstI<br>
 +
AATTC CGTCTC A  GTGG  GCATCAGT  CGAG  A GAGACC CTGCA<br>
 +
    G GCAGAG T  CACC  CGTAGTCA  GCTC  T CTCTGG G
 +
<br><br>
 +
'''New HPP Vector'''
 +
# Use BsaI to free up a new START word and a new FINISH word that are different from any of the Gene words (listed below).
 +
# NheI site in the middle for cloning in any XbaI/SpeI BioBrick fragment, such as the ccdB Death Gene or a reporter expression cassette.
 +
# Flanked by EcoRI and PstI sticky ends.<br><br>
  
−
FINISH_Bot (32 nt)
+
  RI    START  <BsaI  NheI  BsaI>  FINISH PstI<br>
 +
AATTC  GTCC  A GAGACC GCTAGC GGTCTC A  CGAG  CTGCA<br>
 +
    G  CAGG  T CTCTGG CGATCG CCAGAG T  GCTC  G<br><br>
  
−
GGGTCTCTCTCGACTGATGCCCACTGAGACGG
+
'''Specific Words Used So Far'''<br><br>
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NewHPPVec_Top (36 nt)
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START GTCC  GGAC <br>
−
AATTCGTCCAGAGACCGCTAGCGGTCTCACGAGCTGCA
+
FINISH CGAG  CTCG<br>
 +
Half-Edge GTGG  CCAC<br>
 +
GFP GGGT    ACCC<br>
 +
RFP CTGC  GCAG<br>
 +
LacZ CCTG  CAGG<br>
 +
AspC CGTC  GACG<br>
 +
PyrE GGCG  CGCC<br>
 +
ilvE GGGG  CCCC<br>
 +
CAT CGGT  ACCG<br>
 +
TyrB CGCA  TGCG<br>
  
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NewHPPVec_Bot (32 nt)
 
−
GCTCGTGAGACCGCTAGCGGTCTCTGGACG
 
  
−
New START Half Edge
+
Diagram of HPP vector Design: [[Media:MOWestidea.pptx]]
−
1. Use BsaI to free up new START word (GTCC) on 5’ end that allows ligation to new HPP vector.
+
<br>
−
2. Use BsmBI to free up half-edge word (GTGG) on 3’ end that allows ligation to any Gene1 half-edge.
 
−
3. In between these is a J23100 promoter.
 
−
4. Flanked by EcoRI and PstI sticky ends for cloning.
 
−
RI  BsaI>  /START        J23100                    /Half  <BsmBI PstI
 
−
AATTC GGTCTC A GTCC TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC GTGG A GAGACG CTGCA
 
−
    G CCAGAG T CAGG AACTGCCGATCGAGTCAGGATCCATGTCACGATCG CACC T CTCTGC G
 
−
                  /                                        /
 
−
BsaI
 
−
 
−
BsmBI
 
−
 
−
New FINISH Half Edge
 
−
1. Use BsmBI to free up half-edge word (GTGG) on 5’ end that allows ligation to any Gene2 half-edge.
 
−
2. Use BsaI to free up new FINISH word (CGAG) on 3’ end that allows ligation to new tr5f4HPP vector.
 
−
3. In between these is a 8 nt spacer.
 
−
4. Flanked by EcoRI and PstI sticky ends for cloning.
 
−
RI  BsmBI>  /Half  Spacer  /FINISH  <BsaI  PstI
 
−
AATTC CGTCTC A  GTGG  GCATCAGT  CGAG  A GAGACC CTGCA
 
−
    G GCAGAG T  CACC  CGTAGTCA  GCTC  T CTCTGG G
 
−
                                            /                                /
 
−
New HPP Vector
 
−
1. Use BsaI to free up a new START word and a new FINISH word that are different from any of the Gene words (listed below).
 
−
2. NheI site in the middle for cloning in any XbaI/SpeI BioBrick fragment, such as the ccdB Death Gene or a reporter expression cassette.
 
−
3. Flanked by EcoRI and PstI sticky ends.
 
−
START GTCC  GGAC
 
−
FINISH CGAG  CTCG
 
−
Half-Edge GTGG  CCAC
 
−
GFP GGGT    ACCC
 
−
RFP CTGC  GCAG
 
−
LacZ CCTG  CAGG
 
−
AspC CGTC  GACG
 
−
PyrE GGCG  CGCC
 
−
ilvE GGGG  CCCC
 
−
CAT CGGT  ACCG
 
−
TyrB CGCA  TGCG
 
−
  RI  / START    <BsaI    NheI    BsaI>     / FINISH  PstI
 
−
AATTC  GTCC  A GAGACC GCTAGC GGTCTC A  CGAG  CTGCA
 
−
    G  CAGG  T CTCTGG CGATCG CCAGAG T  GCTC  G
 
−
              /                                    /
 

Latest revision as of 20:27, 26 August 2011

New START Half Edge

  1. Use BsaI to free up new START word (GTCC) on 5’ end that allows ligation to new HPP vector.
  2. Use BsmBI to free up half-edge word (GTGG) on 3’ end that allows ligation to any Gene1 half-edge.
  3. In between these is a J23100 promoter.
  4. Flanked by EcoRI and PstI sticky ends for cloning.

  RI   BsaI>   START               J23100               Half   <BsmBI  PstI

AATTC GGTCTC A GTCC TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC GTGG A GAGACG CTGCA

    G CCAGAG T CAGG AACTGCCGATCGAGTCAGGATCCATGTCACGATCG CACC T CTCTGC G



New FINISH Half Edge

  1. Use BsmBI to free up half-edge word (GTGG) on 5’ end that allows ligation to any Gene2 half-edge.
  2. Use BsaI to free up new FINISH word (CGAG) on 3’ end that allows ligation to new tr5f4HPP vector.
  3. In between these is a 8 nt spacer.
  4. Flanked by EcoRI and PstI sticky ends for cloning.

  RI  BsmBI>    Half   Spacer   FINISH   <BsaI  PstI

AATTC CGTCTC A  GTGG  GCATCAGT   CGAG  A GAGACC CTGCA

    G GCAGAG T  CACC  CGTAGTCA   GCTC  T CTCTGG G



New HPP Vector

  1. Use BsaI to free up a new START word and a new FINISH word that are different from any of the Gene words (listed below).
  2. NheI site in the middle for cloning in any XbaI/SpeI BioBrick fragment, such as the ccdB Death Gene or a reporter expression cassette.
  3. Flanked by EcoRI and PstI sticky ends.

 RI    START   <BsaI   NheI   BsaI>   FINISH PstI

AATTC  GTCC  A GAGACC GCTAGC GGTCTC A  CGAG  CTGCA

    G  CAGG  T CTCTGG CGATCG CCAGAG T  GCTC  G

Specific Words Used So Far

START GTCC GGAC
FINISH CGAG CTCG
Half-Edge GTGG CCAC
GFP GGGT ACCC
RFP CTGC GCAG
LacZ CCTG CAGG
AspC CGTC GACG
PyrE GGCG CGCC
ilvE GGGG CCCC
CAT CGGT ACCG
TyrB CGCA TGCG


Diagram of HPP vector Design: Media:MOWestidea.pptx

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