Team:Warsaw/Calendar-Main/30 September 2008

From 2008.igem.org

Revision as of 21:19, 26 October 2008 by MKrzyszton (Talk | contribs)

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
next notebook entry

 


Mutagenesis of protein A

Paweł

Results of sequencing: unfortunately all sequences were wild-type.

Preparation of vectors for Biobricks

Michał K.

  1. Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
  2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmids.

Preparation of BioBricks

Michał K.

    Fig. 1
  1. Digest of pACYC177+OmpA_omega_deltaA_alpha with BamHI and PstI (BamHI buffer) Fig. 2.
  2. Dephosphorylation (CIAP) of plasmids.
  3. Gel elctrophoresis and gel-out of proper band: ??????.
  4. Digest of omega_link fragment (PCR from previous day) with EcoRI and BcuI (BamHI buffer)
  5. Digest of OmpA_omega with BglII and PstI (Orange buffer).
  6. Clean-up of above digest reactions.
  7. Overnight digest of purified pLac_OmpA_omega fragment (from previous day) with EcoRI (EcoRI buffer). DNA ends blunting with Klenow fragment.

Piotr

  1. E. coli TOP10 transformation with overnight ligations pSB1A3 + Z(BBa_K103004) and pSB1A3 + OmpA_link).
  2. Plating on LB + ampicillin.
  3. Inoculation of bacteria received from iGEM HQs, carrying pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
Fig. 1. Control EcoRI/BcuI digests of isolated plasmids
1. Marker
2-3. digested plasmids BBa_I739204
4. digested plasmid psB2K3 Fig. 2. Control BamHI/PstI digests of isolated plasmids
1. Marker
2. digested plasmid pACYC_OmpA_omega_deltaA_alpha