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 purification of Construct 17 parts A and B

• For each PCR, load 10uL of PCR product premixed with 2uL of loading buffer in a single well of a 1% agarose gel
• Cut out the bands, put them into a single 1.5mL microcentrifuge tube
• Add 650uL of ADB Buffer
• Proceed with the Zymo Gel Purification procedure

Zymo Gel Purification clean up procedure of Construct 17 Parts A and B For each of parts A and B:
(All spins until the drying step are 15 second full speed spins)

• cut out bands minimizing extra gel matter.
• put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
• heat at 55, shake and/or vortex until the gel has dissolved.
• If the DNA is <300bp add 250uL of isopropanol
• transfer into the Zymo column inside a collection tube (small clear guys)
• spin through, discard waste.
• Add 200 uL of PE buffer (which is basically 70% ethanol)
• spin through, discard waste.
• Add 200 uL of PE buffer
• spin through, discard waste.
• spin for 90 seconds, full speed to dry.
• elute with 8.5 uL of water into a fresh Eppendorf tube

• Elute the DNA in 50uL of water
• Set up your second round of PCR as a normal 33uL reaction using the eluted mixture of fragments as template

Second PCR reaction of Construct 17

• 24uL ddH2O
• 3.3uL 10x Expand Buffer "2"
• 3.3uL dNTPs (2mM in each)
• 1uL Oligo 1, 10uM
• 1uL Oligo 2, 10uM
• 0.5uL Expand polymerase "1"
• 0.5uL Template DNA

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)