SBB09Ntbk-Dirkvs: Difference between revisions
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| Line 18: | Line 18: | ||
Today, I set up the PCR reactions for the three parts. | Today, I set up the PCR reactions for the three parts. | ||
Dilute oligonucleotides for construct 17 | '''Dilute oligonucleotides for construct 17''' | ||
* Add to the tube a volume of ddH20 10x ul the molar amount of DNA | * Add to the tube a volume of ddH20 10x ul the molar amount of DNA | ||
* Mix and spin | * Mix and spin | ||
| Line 24: | Line 24: | ||
* Mix and spin | * Mix and spin | ||
Prepare the PCR reaction for construct 17 A (Restriction site removal) | '''Prepare the PCR reaction for construct 17 A (Restriction site removal)''' | ||
* 24 uL water | * 24 uL water | ||
* 3.3 uL Expand 10x Buffer 2 | * 3.3 uL Expand 10x Buffer 2 | ||
| Line 32: | Line 32: | ||
* 0.5 uL Expand Polymerase 1 | * 0.5 uL Expand Polymerase 1 | ||
* Run this PCR on program 55 | * Run this PCR on program 55 | ||
** '''FORGOT TO ADD TEMPLATE DNA [[User:Dirk Van Swaay|Dirk]] 16:03, 18 February 2009 (EST)''' | ** '''''FORGOT TO ADD TEMPLATE DNA [[User:Dirk Van Swaay|Dirk]] 16:03, 18 February 2009 (EST)''''' | ||
Prepare the PCR reaction for construct 17 B (Restriction site removal) | '''Prepare the PCR reaction for construct 17 B (Restriction site removal)''' | ||
* 24 uL water | * 24 uL water | ||
* 3.3 uL Expand 10x Buffer 2 | * 3.3 uL Expand 10x Buffer 2 | ||
| Line 42: | Line 42: | ||
* 0.55 uL Expand Polymerase 1 | * 0.55 uL Expand Polymerase 1 | ||
* Run this PCR on program 55 | * Run this PCR on program 55 | ||
** '''FORGOT TO ADD TEMPLATE DNA [[User:Dirk Van Swaay|Dirk]] 16:03, 18 February 2009 (EST)''' | ** '''''FORGOT TO ADD TEMPLATE DNA [[User:Dirk Van Swaay|Dirk]] 16:03, 18 February 2009 (EST)''''' | ||
Dilute oligonucleotides for construct 18 | '''Dilute oligonucleotides for construct 18''' | ||
* Add to the tube a volume of ddH20 10x ul the molar amount of DNA | * Add to the tube a volume of ddH20 10x ul the molar amount of DNA | ||
* Mix and spin | * Mix and spin | ||
Prepare PCR reaction for construct 18 (Wobble) | '''Prepare PCR reaction for construct 18 (Wobble)''' | ||
* 29 uL water | * 29 uL water | ||
* 5 uL Expand 10x Buffer 2 | * 5 uL Expand 10x Buffer 2 | ||
| Line 56: | Line 56: | ||
* 0.75 uL Expand Polymerase 1 | * 0.75 uL Expand Polymerase 1 | ||
Dilute oligonucleotides for construct 19 | '''Dilute oligonucleotides for construct 19''' | ||
* Add to the tube a volume of ddH20 10x ul the molar amount of DNA | * Add to the tube a volume of ddH20 10x ul the molar amount of DNA | ||
* Mix and spin | * Mix and spin | ||
| Line 62: | Line 62: | ||
* Mix and spin | * Mix and spin | ||
Prepare the PCR reaction for construct 19 (Synthesis) | '''Prepare the PCR reaction for construct 19 (Synthesis)''' | ||
* 22 uL water | * 22 uL water | ||
* 3.3 uL Expand 10x Buffer 2 | * 3.3 uL Expand 10x Buffer 2 | ||
Revision as of 14:04, 18 February 2009
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)
- 0.55 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
to do
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)
