M13 redesign, expt'l details: Difference between revisions
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===Vectors=== | ===Vectors=== | ||
[[Image:Macintosh HD-Users-nkuldell-Desktop-M13K07 plasmid-M13K07plasmidmap.jpg|thumb|right|600px| M13K07 plasmid map, from NEB]] | |||
*M13KE [[http://www.westburg.nl/pages/TechnicalResources/Vectors.htm]] | *M13KE [[http://www.westburg.nl/pages/TechnicalResources/Vectors.htm]] | ||
*pUC118 [[http://seq.yeastgenome.org/vectordb/vector_descrip/PUC118.html]] or 119 [[http://seq.yeastgenome.org/vectordb/vector_descrip/PUC119.html]] | *pUC118 [[http://seq.yeastgenome.org/vectordb/vector_descrip/PUC118.html]] or 119 [[http://seq.yeastgenome.org/vectordb/vector_descrip/PUC119.html]] | ||
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===Primers=== | ===Primers=== | ||
====to delete Pst site from M13K07 polylinker | ====For M13K07 manipulations==== | ||
*<b>First need to delete Pst site from M13K07 polylinker</b> | |||
ChangePst2RI_top<br> | these oligos should both destroy Pst and add unique EcoRI to position 8083<br> | ||
*5'ATAG AAT TCT ATG CA | * NOXXX: ChangePst2RI_top<br> | ||
ChangePst2RI_btm <br> | **5'ATAG AAT TCT ATG CA | ||
*5'ACG TTAT CTT AAG AT | * NOXXX: ChangePst2RI_btm <br> | ||
**5'ACG TTAT CTT AAG AT <br> | |||
<br> | |||
AddPst2gVIII_fwd<br> | *<b>Next step is to add PstI to Petrenko site</b> | ||
*CGT TCCGATGCTG TCTTTCGCTG CaGAGGGTGA CGATCCCGCA AAAGCGGCC | use site directed mutagenesis to change GCT to GCA (silent change at Ala, first residue in mature protein)<br> | ||
* Tm 74° | *NOXXX: AddPst2gVIII_fwd<br> | ||
AddPst2gVIII_rev | **CGT TCCGATGCTG TCTTTCGCTG CaGAGGGTGA CGATCCCGCA AAAGCGGCC | ||
*GGC CGC TTT TGC GGG ATC GTC ACC CTC TGC AGC GAA AGA CAG CAT CGG AAC G | ** Tm 74° | ||
*Tm 74° | *NOXXX: AddPst2gVIII_rev | ||
====M13K07 sequencing | **GGC CGC TTT TGC GGG ATC GTC ACC CTC TGC AGC GAA AGA CAG CAT CGG AAC G | ||
M13KO7_seq1100_fwd | **Tm 74° | ||
*TAAGTAACATG GAGCAGGTCG CGGATTTCGA CAC | ====For M13K07 sequencing==== | ||
*Tm 65° | *NOXXX: M13KO7_seq1100_fwd | ||
**TAAGTAACATG GAGCAGGTCG CGGATTTCGA CAC | |||
**Tm 65° | |||
Revision as of 11:18, 26 October 2006
Vectors
Primers
For M13K07 manipulations
- First need to delete Pst site from M13K07 polylinker
these oligos should both destroy Pst and add unique EcoRI to position 8083
- NOXXX: ChangePst2RI_top
- 5'ATAG AAT TCT ATG CA
- NOXXX: ChangePst2RI_btm
- 5'ACG TTAT CTT AAG AT
- 5'ACG TTAT CTT AAG AT
- Next step is to add PstI to Petrenko site
use site directed mutagenesis to change GCT to GCA (silent change at Ala, first residue in mature protein)
- NOXXX: AddPst2gVIII_fwd
- CGT TCCGATGCTG TCTTTCGCTG CaGAGGGTGA CGATCCCGCA AAAGCGGCC
- Tm 74°
- NOXXX: AddPst2gVIII_rev
- GGC CGC TTT TGC GGG ATC GTC ACC CTC TGC AGC GAA AGA CAG CAT CGG AAC G
- Tm 74°
For M13K07 sequencing
- NOXXX: M13KO7_seq1100_fwd
- TAAGTAACATG GAGCAGGTCG CGGATTTCGA CAC
- Tm 65°
