SBB09Ntbk-Dirkvs

Dirk 13:31, 25 February 2009 (EST)

Notes

To do today:
* Analytical clean up, digestion and gel purification of Construct 17
* Finish digestion and cleaning of Construct 18
* Clean up, digest, clean up, and ligation of Construct 19

EcoRI/BamHI Digest of Construct 18 Wobble Product

• Set up the following reaction in a PCR tube:
  • 50uL eluted DNA
  • 5.7uL NEB Buffer 2
  • 1uL EcoRI
  • 1uL BamHI
• Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
• Incubate the reaction at 37 degrees on the thermocycler
• Proceed to another Zymo small fragment cleanup

Second Small-Frag Zymo Cleanup of Construct 18

• Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
• Transfer into the Zymo column (small clear guys)
• Add 500uL of Ethanol and pipette up and down to mix
• spin through, discard waste.
• Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
• spin through, discard waste.
• Add 200 uL of PE or Zymo Wash buffer
• spin through, discard waste.
• spin for 90 seconds, full speed to dry.
• elute with water into a fresh Eppendorf tube

Dirk 13:24, 23 February 2009 (EST)

Summary of activities:
* There was no time to carry out the digestion and second clean up of Construct 18
* Construct 18 is eluted in water in an Eppendorf tube
* Construct 18 had a blue liquid at the bottom of the collection tube after the drying step in the first Zymo Cleanup
* Constructs 17 and 19 have been handed over to Gabriel in three tubes bound by red tape for PCR

To do today:
* Redo the PCR for Construct 17 {<ECOHLY>} (see entry for 18 Feb 2009)
* Cleanup, digest, and cleanup for Construct 18 {<AG4>}
* Run second PCR for Construct 19 {<CBK>}

Construct 17 {<ECOHLY>}

Dilute oligonucleotides for construct 17

• Add 1 ul of oligo dilution to 9 ul of ddH20
• Mix and spin

Prepare the PCR reaction for construct 17 A (Restriction site removal)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007F (10uM)
• 1 uL Oligo Odvs008rR (10uM)
• 0.5 uL Expand Polymerase 1
• 0.5 uL Template DNA E.coli 0157:H7
• Run this PCR on program 55

Prepare the PCR reaction for construct 17 B (Restriction site removal)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007R (10uM)
• 1 uL Oligo Odvs008rF (10uM)
• 0.5 uL Expand Polymerase 1
• 0.5 uLTemplate DNA E.coli 0157:H7
• Run this PCR on program 55

Construct 18 {<AG4>}

Cleanup, digest, and then cleanup again of construct 18.

First Small-Frag Zymo Cleanup of Construct 18

• Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
• Transfer into the Zymo column (small clear guys)
• Add 500uL of Ethanol and pipette up and down to mix
• spin through, discard waste.
• Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
• spin through, discard waste.
• Add 200 uL of PE or Zymo Wash buffer
• spin through, discard waste.
• spin for 90 seconds, full speed to dry.
  • A small amount of blue liquid was present in the collection tube at the end of this step. Dirk 15:38, 23 February 2009 (EST)
• elute with 50 ul water into a fresh Eppendorf tube

Construct 19 {<CBK>}

Dilute oligonucleotides for construct 19

• Add 1 ul of oligo dilution to 9 ul of ddH20
• Mix and spin

Prepare the second PCR reaction for construct 19 (Synthesis)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs003 (10uM)
• 1 uL Oligo Odvs006 (10uM)
• 0.5 uL Expand Polymerase 1
• 0.5 uL Template DNA
• Run this PCR on program 55

Dirk 13:21, 18 February 2009 (EST)

Today, I set up the PCR reactions for the three parts.

Dilute oligonucleotides for construct 17

• Add to the tube a volume of ddH20 10x ul the molar amount of DNA
• Mix and spin
• Add 1 ul of oligo dilution to 9 ul of ddH20
• Mix and spin

Prepare the PCR reaction for construct 17 A (Restriction site removal)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007F (10uM)
• 1 uL Oligo Odvs008rR (10uM)
• 0.5 uL Expand Polymerase 1
• Run this PCR on program 55
  • FORGOT TO ADD TEMPLATE DNA Dirk 16:03, 18 February 2009 (EST)

Prepare the PCR reaction for construct 17 B (Restriction site removal)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007R (10uM)
• 1 uL Oligo Odvs008rF (10uM)
• 0.5 uL Expand Polymerase 1
• Run this PCR on program 55
  • FORGOT TO ADD TEMPLATE DNA Dirk 16:03, 18 February 2009 (EST)

Dilute oligonucleotides for construct 18

• Add to the tube a volume of ddH20 10x ul the molar amount of DNA
• Mix and spin

Prepare PCR reaction for construct 18 (Wobble)

• 29 uL water
• 5 uL Expand 10x Buffer 2
• 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 5 uL Oligo Odvs002F (100uM)
• 5 uL Oligo Odvs002R (100uM)
• 0.75 uL Expand Polymerase 1

Dilute oligonucleotides for construct 19

• Add to the tube a volume of ddH20 10x ul the molar amount of DNA
• Mix and spin
• Add 1 ul of oligo dilution to 9 ul of ddH20
• Mix and spin

Prepare the PCR reaction for construct 19 (Synthesis)

• 22 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs003 (10uM)
• 1 uL Oligo Odvs004 (10uM)
• 1 uL Oligo Odvs005 (10uM)
• 1 uL Oligo Odvs006 (10uM)
• 0.5 uL Expand Polymerase 1
• Run this PCR on program 55

Will have to redo construct 17 because I forgot to add the template DNA to the reactions.

Dirk 00:39, 10 February 2009 (EST)

Carried out the oligos and construction file for the Cellulose Binding Knottin CBK (Bdvs003, or M10046).

• Construction file to be found here (M10046)
• Oligonucleotide sequences to be found here (M10046)

Dirk 00:17, 10 February 2009 (EST)

Carried out the oligos and construction file for the Silver-binding peptide AG4 (Bdvs002, or M10023).

• Construction file to be found here (M10023)
• Oligonucleotide sequences to be found here (M10023)

* Design Oligos and make construction file for Cellulse Binding Knottin (M10046)

Dirk 15:01, 9 February 2009 (EST)

Got the project ball on Wednesday last week. Assigned project 15954 - Beta Roll and Silver Peptide. Also assigned Cellulose Binding Knottin, from 37738. The project page can be found here (SBB09_15954).

Spent the lab time designing the oligos and writing the construction file for the Beta Roll (Bdvs001, or M10022).

• Construction file to be found here (M10022)
• Oligonucleotide sequences to be found here (M10022)

* Design oligos and make construction file for Silver Peptide (M10023)
* Design Oligos and make construction file for Cellulse Binding Knottin (M10046)