SBB09Ntbk-Dirkvs: Difference between revisions
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<pre> | <pre> | ||
Summary of Activities: | Summary of Activities: | ||
* Transformation of Construct 17 looked pretty decent - Tim grew up 4 clones for miniprep and | * Transformation of Construct 17 looked pretty decent - Tim grew up 4 clones for miniprep and mapping | ||
* Miniprep of Construct 17 carried out by CA | |||
* Construct 18 colonies didn't grow. The construct was abandoned (Although CA might try to rescue it) | * Construct 18 colonies didn't grow. The construct was abandoned (Although CA might try to rescue it) | ||
* Ligation of Construct 19 into pBca9495AK was carried out | * Ligation of Construct 19 into pBca9495AK was carried out |
Revision as of 12:11, 6 March 2009
Dirk 13:13, 6 March 2009 (EST)
Summary of Activities: * Transformation of Construct 17 looked pretty decent - Tim grew up 4 clones for miniprep and mapping * Miniprep of Construct 17 carried out by CA * Construct 18 colonies didn't grow. The construct was abandoned (Although CA might try to rescue it) * Ligation of Construct 19 into pBca9495AK was carried out * Construct 19 GP CLEAN (pre-ligation) stored in blue-stripes box with less than 10 ul volume * To do today: * Miniprep and map of Construct 17 * Ligation (into pBca9495AK), transformation, and plating of Construct 19
Construct 17 {<ECOHLY>}
Miniprep purification of Construct 17 in pDrive DNA - Carried out by CA ---Dirk 14:09, 6 March 2009 (EST)
- MINIPREP (1mL - 5mL) Procedure for Plasmid DNA Purification (using the QIAGEN QIAPrep Spin Miniprep kit)
- Pellet around 1.5 mL or 2 mL saturated culture by spinning full speed, 30 seconds.
- Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL)
- Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
- Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
- Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
- Spin in centrifuge at top speed for 5 minutes.
- Label blue columns with an alcohol-resistant lab pen.
- Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
- Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
- Wash each column with 500 uL of PB buffer.
- Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
- Wash with 750uL of PE buffer (washes the salts off the resins).
- Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
- Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
- Label new tubes and put columns in them.
- Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
- Spin in centrifuge at top speed for 30 seconds.
- Take out columns and cap the tubes.
- Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.
Construct 18 {<AG4>}
Construct 18 was abandoned - the colonies didn't grow, and it is too late to start over. Dirk 13:26, 6 March 2009 (EST)
Construct 19 {<CBK>}
Ligation (into pBca9495AK), transformation, and plating
Ligation of Construct 19 EcoRI/BamHI digests into pBca9495AK
- Set up the following reaction:
6.5uL ddH2O 1uL T4 DNA Ligase Buffer (small red or black-striped tubes) 1uL Vector digest 1uL Insert digest 0.5uL T4 DNA Ligase
- Pound upside down on the bench to mix
- Give it a quick spin to send it back to the bottom of the tube
- Incubate on the benchtop for 30min
- Put on ice and proceed to the transformation
Transformation of Construct 19 and pBca9495AK cloning vector by heat-shock
- Put your ligation mixture on ice, let it cool a minute or two
- Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
- Let sit on ice for 10 min
- Heat shock for 2 min at 42
- Put back on ice for 1 min
- For ampicillin selection, you can plate immediately, otherwise:
- Add 100uL of 2YT, let shake in the 37 degree incubator for 60 min
Dirk 17:04, 4 March 2009 (EST)
Summary of Activities: * Construct 17 cells were plated on AMP dish and left to incubate in room 327 * Construct 18 cells were plated on AK dish and left to incubate in room 327 To do today: * Plate Construct 17 cells on AMP dish * Plate Construct 18 cells on AK dish
Construct 17 {<ECOHLY>}
Plate Construct 17 cells on AMP dish
- Plate on selective antibiotics, let incubate overnight
Construct 18 {<AG4>}
Plate Construct 18 cells on AK dish
- Plate on selective antibiotics, let incubate overnight
Dirk 13:18, 4 March 2009 (EST)
Summary of Activities: * Carried out ligation of Construct 17 into vector pDrive * Carried out transformation of Construct 17 - must return in 40 mins to plate cells * Construct 17 Broll PCR PROD stored in blue-striped box * Carried out ligation of Construct 18 into vector pBca9495AK * Carried out transformation of Construct 18 - must return in 40 mins to plate cells * Construct 18 DIGESTED CLEAN stored in blue-striped box with 49 ul volume * Carried out gel purification of Construct 19 - bands were difficult to see * Carried out GP Zymo Cleanup of Construct 19 GP product * Did not carry out ligation of Construct 19 * Construct 19 GP CLEAN stored in blue-stripes box with 8.5 ul volume To do today: * Ligation of Construct 17 in preparation for cloning and sequencing, before making into a part * Ligation of Construct 18 * Gel purification and ligation of Construct 19
Construct 17 {<ECOHLY>}
Ligation of Construct 17 PCR product into pDrive Cloning Vector
- Add 1 ul of pDrive cloning vector (50 ng/ul)
- Add 1 ul of Construct 17 PCR product
- Add 3 ul of RNase-free water
- Add 5 ul of Ligation Master Mix, 2x
- Mix the ligation reaction and incubate for 30 min at room temperature
Transformation of Construct 17 and pDrive cloning vector by heat-shock
- Put your ligation mixture on ice, let it cool a minute or two
- Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
- Let sit on ice for 10 min
- Heat shock for 2 min at 42
- Put back on ice for 1 min
- For ampicillin selection, you can plate immediately, otherwise:
- Add 100uL of 2YT, let shake in the 37 degree incubator for 60 min
Construct 18 {<AG4>}
Ligation of Construct 18 EcoRI/BamHI digests into pBca9495AK
- Set up the following reaction:
6.5uL ddH2O 1uL T4 DNA Ligase Buffer (small red or black-striped tubes) 1uL Vector digest 1uL Insert digest 0.5uL T4 DNA Ligase
- Pound upside down on the bench to mix
- Give it a quick spin to send it back to the bottom of the tube
- Incubate on the benchtop for 30min
- Put on ice and proceed to the transformation
Transformation of Construct 18 and pBca9495AK cloning vector by heat-shock
- Put your ligation mixture on ice, let it cool a minute or two
- Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
- Let sit on ice for 10 min
- Heat shock for 2 min at 42
- Put back on ice for 1 min
- For ampicillin selection, you can plate immediately, otherwise:
- Add 100uL of 2YT, let shake in the 37 degree incubator for 60 min
Construct 19 {<CBK>}
Gel purification of Construct 19 digestion product
- Load 10uL of digestion 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 of Construct 19 digestion gel purification product
- 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
Dirk 13:18, 2 March 2009 (EST)
Summary of Activities: * Construct 17 PCR reactions worked, as verified by Chris Anderson * New oligos for a second restriction site in Construct 17 were ordered * No work on Construct 17 was carried out today * First Zymo small fragment clean up of Construct 18 wobble product carried out * Digestion of Construct 18 was carried out (with full wobble product volume - 50 ul) * Second Zymo small fragment clean up of Construct 18 digestion product carried out * Construct 18 DIGESTED CLEAN stored in blue-stripes box with 50 ul * Digestion of Construct 19 was carried out (with 8 ul of gene synthesis reaction volume) * Small fragment Zymo clean up of Construct 19 digestion product carried out * Gel purification of Construct 19 digestion product not carried out * Analytical gel of Construct 19 pre-digestion was carried out - part was there * Construct 19 DIGESTED CLEAN NOGP stored in blue-stripes box with 10 ul * Construct 19 PRE DIGEST stored in blue-stripes box with 32 ul left To do today: * Clean, digest, clean, and ligation of Construct 18 * Analytical gel of Construct 19 second PCR product * Digest, clean, gel purification, and ligation of Construct 19
Construct 18 {<AG4>}
Clean, digest, clean, and ligation 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.
- elute with water into a fresh Eppendorf tube
EcoRI/BamHI Digest of Construct 18 Wobble Product
For wobble products, you will digest the entire extension reaction-worth of DNA. Since you started with 50uL of extension reaction, you should now have 50uL of eluted DNA in water.
- 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
Construct 19 {<CBK>}
Digest, clean, gel purification, and ligation of Construct 19
EcoRI/BamHI Digest of Construct 19 PCR Products
For PCR products, you will only digest a portion of your purified PCR product. Note that you must make a minor modification to the procedure if your DNA is shorter than 300bp (you add a little isopropanol to the ADB after melting).
- 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
- 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
- If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column
Analytical Gel of Construct 19 second PCR product
- Load 3uL of digestion product premixed with 1uL of loading buffer in a single well of a 1% agarose gel
- Results shown to the right
Small-Frag Zymo Cleanup of Construct 19 digestion product for storage before Gel Purification
- 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 10ul water into a fresh Eppendorf tube
Dirk 15:24, 27 February 2009 (EST) Starting over with Construct 17
Summary of Activities: * Fixed errors in construction file * Found out the correct template DNA (CFT03) * Started the PCR reaction for Construct 17 A, B, and C using CFT03 as template ** A is the strand upstream from the restriction site (379 bp) ** B is the downstream strand from the restriction site (89 bp) ** C is the full part without restriction site removal (436 bp) * Need to find out what the exact sequence will be for each of these PCR products To do today: * First PCR of Construct 17, making parts A and B (second attempt)
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
Prepare the PCR reaction for construct 17 C (Straight forward PCR - no 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
Dirk 13:13, 27 February 2009 (EST)
Summary of Activities: * Gel purification of Construct 17 parts A and B was attempted - parts weren't there: either PCR failed, or DNA degraded * Construct 17 parts A and B (pre GP) stored in blue-stripes box, bound in red tape * Restarted the work with Construct 18 * Wobble PCR reaction for Construct 18 was prepared and given in red tape * Discarded the Construct 19 (IPA ERROR) container * No more work was carried out with Construct 19 today (no digestion) * Construct 19 (PRE DIGEST) stored in blue-stripes box 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 (Do GP afterwards - ie, normal PCR 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
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)
- The bottle was mislabelled - used 10 mM dNTPs instead. Dirk 15:06, 27 February 2009 (EST)
- 5 uL Oligo Odvs002F (100uM)
- 5 uL Oligo Odvs002R (100uM)
- 0.75 uL Expand Polymerase 1
Construct 19 {<CBK>}
No work was carried out with Construct 19 today.
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)