SBB10Ntbk-JingLuo
Inventory (Inside Box C)
- y A(star) - part A for SOEing
- y B(star) - part B for SOEing
- y C(star) - part C for SOEing
- JHL Gel frag - preparative gel fragments of PCR A, B, C dissolved in ADB buffer
- PCR Zc ABC - Zymo gel clean preparative gel fragments of PCR A, B, C
- sbb20 SOE H2O - 2nd round of SOEing of PCR products A, B, C
- sb20 digest zc - Digested w/ EcoR1/BamH1 and zymo cleaned of the Round 2 SOEing reaction
- 20 digest prep gel - sbb20 from preparative gel after digestion
- JHL SOE Pro Zc -
- sbb20 Sew - SOEing with external primers, first attempt
- Xfer digest zc - Eco/Bam transfer digest and zymo cleaned
- MP20 #1 - Miniprepped sbb20 colony from transformed cells w/ part
- MP20 #2 - Miniprepped sbb20 colony from transformed cells w/ part
- MP20 #3 - Miniprepped sbb20 colony from transformed cells w/ part
- MP20 #4 - Miniprepped sbb20 colony from transformed cells w/ part
- sb35 1 - Miniprepped sbb35 colony from transformed cells w/ part
- sb35 2 - Miniprepped sbb35 colony from transformed cells w/ part
- sb35 3 - Miniprepped sbb35 colony from transformed cells w/ part
- sb35 4 - Miniprepped sbb35 colony from transformed cells w/ part
2/17
Part sbb20:
Purpose: Using the designed oligos to clone the fragments of sbb20, which will later be SOE'd together.
Protocol:
- Using the 2K55 temperature program, cloned the necessary parts for SOEing using the protocol: Cloning by PCR
Results
- The next step is to run a preparative gel to determine the results of SOEing.
Products labeled:
- y A(star) (sbb20A), y B(star) (sbb20B), y C(star) (sbb20C)
2/22
Part sbb20:
Purpose: Purifying the DNA from the PCR reactants and any unwanted PCR products.
Protocol:
- Ran a preparative gel on the PCR products from 2/17 (sbb20A,sbb20B,sbb20C)
- Ran a gel purification of the preparative gel fragments using protocol: Zymo gel purification
Results
- Results of the preparative gel looked good. Lanes 9, 10, 11 (Part A, B, C respectively): Gel pic
Products labeled:
- Preparative gel: JHL Gel frag
- Post zymo clean: JHL Gel pure ZC or PCR Zc ABC
2/24
Part sbb20:
Purpose:
Protocol:
- Ran a SOEing reaction using protocol: SOEing by PCR
Labeled: SOE JHL
Results
3/1
Part sbb20:
Purpose:
Protocol:
- Ran an analytical gel
- Results of analytical gel showed SOEing PCR failed to produce any product: Gel pic
- Repeated SOEing PCR modified to use DMSO and temperature program 45: SOEing by PCR
Results
3/3
Part sbb20:
Purpose:
Protocol:
- Ran on preparative gel.
- Extracted the gel and ran a gel purification using protocol: Zymo gel purification.
- Digested the product with restriction enzymes using the protocol: EcoR1/BamH1 Digest of PCR products
Results
3/8
Part sbb20:
Purpose:
Protocol:
- Ligated the part with the pBjk2741 vector using protocol: Ligation of EcoR1/BamH1 Digests
- Transformed into JTK086 spec cells using protocol: Transformation by Heat Shock
- GSI's plated out onto spec antibiotic plate.
Results
Part sbb35:
Purpose:
Protocol:
- Began a Eco/Bam transfer using protocol: EcoR1/BamH1 Part Transfer
- stopped after the restriction enzyme digestion that was done using protocol: EcoR1/BamH1 Digest of PCR products
- performed a regular zymo clean up on the part using protocol: Regular zymo cleanup
Results
3/10
Part sbb20:
Purpose:
Protocol:
- GSI's picked out colonies and incubated them using protocol: Picking of Colonies
- Minipepped cells using protocol: Macherey-Nagel Minipepped
Results
Part sbb35:
Purpose:
Protocol:
- Ligated the part with pBca1256 vector instead of pBca9523 using protocol: Ligation of EcoR1/BamH1 Digests
- Transformed into DH108 spec cells using protocol: Transformation by Heat Shock
- Plated out onto spec antibiotic plate
Results
3/15
Part sbb20:
Purpose:
Protocol:
- Ran an analytical digest map using protocol: Analytical digest mapping
- Results of the analytical digest: Gel pic
Results
Part sbb35:
Purpose:
Protocol:
- Picked colonies and miniprepped the cells using protocol: Macherey-Nagel Minipepped
Results
3/17
Part sbb20:
Purpose:
Protocol:
- Sequencing
Results
Part sbb35:
Purpose:
Protocol:
- Ran an analytical digest map using protocol: Analytical digest mapping
Results