SBB10Ntbk-JingLuo

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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: Create sbb20 insert -- 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: Create sbb20 insert -- Identify the presence or not of the appropriate sequence, extracting it and purifying it from the gel.
Protocol:

  • Ran a preparative gel on the PCR products from 2/17 (sbb20A,sbb20B,sbb20C)

Results

  • Results of the preparative gel looked good. Lanes 9, 10, 11 (Part A, B, C respectively): Gel pic
  • Ran a gel purification of the preparative gel fragments using protocol: Zymo gel purification

Products labeled:
- Preparative gel: JHL Gel frag
- Post zymo clean: JHL Gel pure ZC or PCR Zc ABC

2/24

Part sbb20:
Purpose: Create sbb20 insert -- Using the external primers to combine the three parts together into sbb20.
Protocol:

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: Create sbb20 insert -- 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: Create sbb20 insert -- Identify the presence or not of the appropriate sequence from the SOEing reactions and extract it.
Protocol:

  • Ran on preparative gel.

Results

Products labeled:
- 20 digest prep gel

3/8

Part sbb20:
Purpose: Create the full correct plasmid (sbb20 part + vector) -- Combine the digested insert (sbb20 part) with the vector, transform into cells, and plate.
Protocol:

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:

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

3/9

Part sbb20:
Results:

  • Colonies did grow and the GSI's picked out colonies and cultured them using protocol: Picking of Colonies

3/10

Part sbb20:
Purpose: Create the full correct plasmid (sbb20 part + vector) -- Extract the DNA from the colonies so that it can be analyzed.
Protocol:

Results

  • Need to determine whether or not the cells successfully took the full plasmid, this will be done next time by analytical gel.

Products labeled:
- MP20 #1, MP20 #2, MP20 #3, and MP20 #4

Part sbb35:
Purpose: Create the full correct plasmid (sbb35 part + vector) -- Combine the insert with the vector, transform into cells, and plate.
Protocol:

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 sbb35

3/11-15

Part sbb35:

3/15

Part sbb20:
Purpose: Create the full correct plasmid (sbb20 part + vector) -- Determine if the transformed cells DNA contain the full plasmid.
Protocol:

Results

  • Results of the analytical digest showed that it succeeded. The cells have an approximately full length plasmid: Gel pic
  • The next step is to sequence the plasmid to make sure that it is 100% what we wanted and no mutations.

Part sbb35:
Purpose: Create the full correct plasmid (sbb35 part + vector) -- Extract and purify the DNA from the cells
Protocol:

Results

  • The next step is to run the DNA on an analytical gel, this will allow us to see roughly whether or not the full sequence is present.

Products labeled:
- sb35 1, sb35 2, sb35 3, sb35 4

3/17

Part sbb20:
Purpose: Create the full correct plasmid (sbb20 part + vector) -- Check sequence of plasmid to target plasmid.
Protocol:

  • Sequencing

Results

  • Will receive the results later.

Products labeled:
- Took the B6 well for sequencing of the sbb20

Part sbb35:
Purpose: Create the full correct plasmid (sbb35 part + vector) -- Determine if the full sequence is present.
Protocol:

Results

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