Orientation Test

1
Orientation Test
PCR Product
T7
EcoRI
EcoRI
PmeI
NotI
PstI
SpeI
T7
Cloning Site
pCR4-TOPO
2
EcoRI
EcoRI
PmeI
NotI
PstI
SpeI
PCR Product
T7
T7
Effective Orientation
EcoRI
EcoRI
PmeI
NotI
PstI
SpeI
PCR Product
T7
T7
Ineffective Orientation
3
SpeI
EcoRI
EcoRI
PmeI
NotI
PstI
SpeI
PCR Product
T7
T7
Vector (4000bp) Small PCR Frag
Digest SpeI
Effective Orientation
SpeI
EcoRI
EcoRI
PmeI
NotI
PstI
SpeI
PCR Product
T7
T7
Vector (4000bp) Larger PCR Frag
Digest SpeI
Ineffective Orientation
4
HT115 DE3

• E. coli Strain
• Null mutation of rnc null (RNaseIII)
• RNase III degrades double stranded RNA
• DE3 lambda lysogen
• containing IPTG inducible T7 RNA polymerase
• Lysogen contains tet resistance gene

Timmons L, Court DL, Fire A (2003)
5
Transformation

• Wednesday
• Add Tetracyline to LB broth
• Inoculate with culture of HT115 DE3
• Grow O/N 37
• Thursday AM
• Prepare second flask of LBtet
• Inoculate with 500ml of overnight culture
  HT115DE3.
• Grow 4 hours
• Thursday PM
• Treat Cells with CaCl to make competent
• Mix cells with RNAi vector
• Spread on LB tet amp plates
• Grow O/N 37