Team:Warsaw/Calendar-Main/13 October 2008

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<var><b>Fig. 1.</b>SacI/BcuI digests of PCR to obtain alpha_linker_2 and linker_alpha<br>
<var><b>Fig. 1.</b>SacI/BcuI digests of PCR to obtain alpha_linker_2 and linker_alpha<br>
1. Marker<br>
1. Marker<br>
-
2. digested PCR product - alpha_linker<br>
+
2. Digested PCR product - alpha_linker<br>
-
3. digested PCR product - linker_alpha<br></var>
+
3. Digested PCR product - linker_alpha<br></var>

Latest revision as of 17:59, 28 October 2008

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Preparation of linker_alpha (BBa_K103009)

Michał K.

  1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using LinLSXNE and AlphaPSpe primers (annealing temperature 58 °C; elongation length 60s) to obtain linker_alpha (BBa_K103009) fragment.
  2. Gel electrophoresis and gel-out of proper band 600 bp - linker_alpha (BBa_K103009) PCR product. Fig. 1.
  3. Digest of isolated PCR product linker_alpha (BBa_K103009) with EcoRI and BcuI (BamHI buffer).
  4. Clean-up of digested PCR product.
  5. Overnight ligation of DNA fragments: pSB2K3 + linker_alpha (BBa_K103009).

Fig. 1.SacI/BcuI digests of PCR to obtain alpha_linker_2 and linker_alpha
1. Marker
2. digested PCR product - alpha_linker
3. digested PCR product - linker_alpha

Preparation of OmpA-linker-omega-linker (BBa_K103016)

Michał K.

Inoculation of few more colonies from old plate with ligation of pACYC177 + OmpA-linker-omega-linker (BBa_K103016) to liquid LB + kanamycin.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K.

Inoculation of colonies from plate with ligation of pSB1A3 carrying alpha_linker under PT7 (BBa_K103019) fragment to liquid LB + ampicillin.

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

Inoculation of colonies from plate with ligation of pSB2K3 + omega_linker under PT7 (BBa_K103020) to liquid LB + kanamycin.

Preparation of AID under pBAD/araC (BBa_K103002)

Michał K.

Overnight ligation of DNA fragments: pSB1A3 (from 6 October) + AID under pBAD/araC (BBa_K103002) (from 10 October).

Preparation of OmpA_linker_alpha_linker under Plac (BBa_K103017)

Michał K.

  1. Digest of pSB2K3 + OmpA_linker_omega_linker under Plac (BBa_K103018) with BcuI and SacI (SacI buffer), dephosphorylation with CIAP.
  2. PCR on alpha PCR product from 10 October using AlphaL+SacI and LinP_BS2 primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha2 fragment with proper restriction sites (SacI and BcuI).
  3. Gel electrophoresis and gel-out of proper bands (600 bp for alpha2 PCR product and 3000 bp for pSB2K3 (with OmpA_linker under PLac promoter) fragment.
  4. Digest of isolated PCR product (alpha2 fragment) with SacI and BcuI (SacI buffer).
  5. Clean-up of digested PCR product. Fig. 1
  6. Overnight ligation of DNA fragments: pSB2K3 (with lactose promoter and OmpA_linker) + alpha2.
Fig. 1.SacI/BcuI digests of PCR to obtain alpha_linker_2 and linker_alpha
1. Marker
2. Digested PCR product - alpha_linker
3. Digested PCR product - linker_alpha