SBB09Ntbk-Dirkvs: Difference between revisions
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=== '''Construct 17 {<ECOHLY>}''' === | === '''Construct 17 {<ECOHLY>}''' === | ||
'''Gel purification of Construct 17 parts A and B''' | |||
*For each PCR, load 8uL of PCR product premixed with 1uL 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 | |||
*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''' | '''Second PCR reaction of Construct 17''' |
Revision as of 12:07, 27 February 2009
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 8uL of PCR product premixed with 1uL 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
- 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
Construct 19 {<CBK>}
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)