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 digested 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: Transfer the part from one vector to another, starting with digestion of the sbb35 part.
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

• The next step is to digest the vector and ligate the two together, whether or not this works will be determined once the cells are miniprepped and a analytical digest is run.

Products labeled:
- Xfer digest zc

3/10

Part sbb20:
Purpose: Extract the DNA from the colonies so that it can be analyzed.
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