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 Sew - SOEing with external primers, first attempt
• 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 -

• 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 the PCR.

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: Using the external primers to combine the three parts together into sbb20.
Protocol:

• Ran a SOEing reaction using protocol: SOEing by PCR
  

Results

• Next step is to run an analytical gel to determine the results of the SOEing reaction.

Products labeled:
- SOE JHL or sbb20 sew

3/1

Part sbb20:
Purpose: Identify whether or not the SOEing reaction produced the full length sequence.
Protocol:

• Ran an analytical gel

Results

• Results of analytical gel showed SOEing PCR failed to produce any product: Gel pic
• Reattempting the SOEing, by repeating with a modified SOEing PCR to use DMSO or H2O and temperature program 45: SOEing by PCR

Products labeled:
- sbb20 SOE H2O
- sbb20 SOE DMSO

3/3

Part sbb20:
Purpose: Identify the presence or not of the appropriate sequence from the SOEing reactions and extract it.
Protocol:

• Ran on preparative gel.

Results

• The full length sequence was present. It was extracted and run through gel purification using protocol: Zymo gel purification.
• Digested the product with restriction enzymes using the protocol: EcoR1/BamH1 Digest of PCR products

Products labeled:
- 20 digest prep gel

3/8

Part sbb20:
Purpose: Combine the insert (sbb20 part) with the vector, transform into cells, and culture.
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 The next step is to see if any colonies grow up on the selective resistance plates, if so pick colonies and culture them. Products labeled:
- JHL sbb20 plate

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