SBB09Ntbk-Dirkvs

Dirk 13:13, 27 February 2009 (EST)

Summary of Activities:
* Restarted the work with Construct 18
* Discarded the Construct 19 (IPA ERROR) container



To do today:
* Second PCR of Construct 17
* Prepare Wobble PCR reactions of Construct 18 (STARTING OVER)
* Clean up, digestion, clean up, and ligation of construct 19 PRE DIGEST

Construct 17 {<ECOHLY>}

Gel analysis, clean up, digestion and gel purification of Construct 17

Gel analysis of Construct 17 SOE product

Zymo clean up of Construct 17 SOE product

Digestion of Construct 17 SOE product

Gel purification of Construct 17

Construct 18 {<AG4>}

Prepare PCR reaction for construct 18 (Wobble) (Further dilutions not necessary)

• 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

Dirk 13:31, 25 February 2009 (EST)

Summary of Activities:
* Second PCR of Construct 17 was not carried out
* First PCR products A+B stored in box, bound in red tape
* Carried out the digestion and second cleaning of Construct 18
* Forgot to add the ADB buffer to the second cleaning of Construct 18 - DNA might be lost
* 50 ul eluted DNA Construct 18 - MAYBE LOST - stored in box
* Did first clean up and digest of Construct 19
* IPA was incorrectly added to the Construct 19 digested DNA - a small fragment clean up was carried out to correct for this mistake
* Gel purification of Construct 19 was not carried out
* 40 ul eluted DNA Construct 19 - PRE DIGESTION - stored in box
* 50 ul eluted DNA Construct 19 - IPA ERROR - stored in box (note that this is diluted 5x)
* Second clean up and ligation of Construct 19 still to be carried out

To do today:
* Second PCR of Construct 17 (Correction: this was not an SOE reaction)
* Finish digestion and cleaning of Construct 18
* Clean up, digest, clean up, and ligation of Construct 19

Construct 17 {<ECOHLY>}

Nothing was carried out with construct 17 today.

Construct 18 {<AG4>}

Digest, and then cleanup again of construct 18.

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 for 1 hour
• 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.
  • Forgot to put the ADB buffer!!! Dirk 15:14, 25 February 2009 (EST)
• 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

Construct 19 {<CBK>}

Clean up and digest of Construct 19

First Small-Frag Zymo Cleanup of Construct 19

• 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.
  • found out what the blue liquid in the previous wash (construct 18) was: the ethanol dissolved some of the marker ink. Dirk 14:35, 25 February 2009 (EST)
• spin for 90 seconds, full speed to dry.
• elute with 50 ul water into a fresh Eppendorf tube

EcoRI/BamHI Digestion of Construct 19 synthesis product

• Set up the following reaction:
  • 8uL of eluted PCR product
  • 1uL of NEB Buffer 2
  • 0.5uL EcoRI
  • 0.5uL BamHI
• Incubate at 37 degrees on the thermocycler for 1hr
  • Incubation carried out for only 40 minutes Dirk 15:27, 25 February 2009 (EST)
• Add 250uL of isopropanol and mix
  • This was incorrect - now, instead of gel purification, a small fragment clean up will be carried out. Dirk 15:38, 25 February 2009 (EST)
• Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point
  • This step was not carried out because of the incorrect addition of IPA to the mixture prior to running the gel. Small fragment clean up carried out instead. Dirk 15:38, 25 February 2009 (EST)

Emergency Small-Frag Zymo Cleanup of Construct 19

• Transfer DNA and IPA mix into the Zymo column (small clear guys)
• Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
  • Solution went dark yellow upon addition of ADB buffer. Dirk 15:45, 25 February 2009 (EST)
• 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 50 ul 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)