Difference between revisions of "GRNA Production"
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(Created page with "# Oligo Rehydration 100 μmol [ ] (stock) of oligos Ex. 100 μmol [ ] —> 27.8 nmol [ ] —> rehydrate oligo bead with 278 μl of H2O *add H2O to hydrate oligos *incubate 5 m...") |
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| − | + | (1) Oligo Rehydration | |
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100 μmol [ ] (stock) of oligos | 100 μmol [ ] (stock) of oligos | ||
| + | |||
Ex. 100 μmol [ ] —> 27.8 nmol [ ] —> rehydrate oligo bead with 278 μl of H2O | Ex. 100 μmol [ ] —> 27.8 nmol [ ] —> rehydrate oligo bead with 278 μl of H2O | ||
| + | |||
*add H2O to hydrate oligos | *add H2O to hydrate oligos | ||
*incubate 5 min at room temp | *incubate 5 min at room temp | ||
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*incubate another 5 min to fully dissolve store at -20°C | *incubate another 5 min to fully dissolve store at -20°C | ||
| − | + | ||
| + | (2) Sense / Antisense Oligo Annealation | ||
TRIS Buffer | TRIS Buffer | ||
| Line 23: | Line 27: | ||
* incubate samples at high temp to low temp (about -1°C / sec) - must reach 95°C | * incubate samples at high temp to low temp (about -1°C / sec) - must reach 95°C | ||
* store at 4°C | * store at 4°C | ||
| + | |||
| + | |||
| + | (3) Plasmid Digestion | ||
| + | |||
| + | Plasmid DR274 - digest with restriction enzyme BsaI | ||
| + | |||
| + | Always mix then centrifuge reaction components after removing from -20° | ||
| + | |||
| + | Reaction Mix (Add in parenthetical order.) | ||
| + | |||
| + | # 14.5μl H2O | ||
| + | # 1μl plasmid DNA (1-2 μg) (1.419 μg/μl —> just use 1 μl) | ||
| + | # 2μl Buffer IV (10x) | ||
| + | # 2μl BSA (10x) | ||
| + | # 10U/μl BsaI (0.5 μl for example) | ||
| + | —————————— | ||
| + | 20 μl final volume | ||
| + | |||
| + | |||
| + | (4) Gel- Plasmid Digestion | ||
| + | |||
| + | Gel Digestion | ||
| + | |||
| + | *1.5% agarose gel | ||
| + | *20 μl digestion vol + 3 μl loading dye | ||
| + | *run for 40 min @ 130 V | ||
| + | *take picture quickly! (avoid excessive UV exposure to nucleic acid) | ||
| + | |||
| + | |||
| + | (5) Gel- DNA Extration/Purification of Plasmid | ||
| + | |||
| + | DNA Extraction Kit (Gel Extraction) | ||
| + | weigh eppendorf mct tube — slice out band from gel | ||
| + | *place in tube | ||
| + | *weigh again | ||
| + | *add volume of Buffer 1 equivalent to weight difference (gel slice weight) — incubate at 60°C for 5-10 min to let gel dissolve in extraction buffer | ||
| + | (should look yellow. pink/red means too basic) | ||
| + | *apply solution to column | ||
| + | *incubate for 1 min | ||
| + | *centrifuge @ 13,000 rpm, 1 min | ||
| + | *put column back to the original tube | ||
| + | *add 500 μl wash B (salt + EtOH) | ||
| + | *centrifuge 13,000 rpm 1-1.5 min | ||
| + | *put column back in column tube | ||
| + | *centrifuge @ 13,000 rpm, 2 min | ||
| + | *put column into new 1.5 mL Eppendorf microcentrifuge tube — add 20ul H2O to column | ||
| + | *incubate 1 min | ||
| + | *centrifuge @ 13,000 rpm, 1 min | ||
| + | *add 20ul H2O to column | ||
| + | *incubate 1 min | ||
| + | *centrifuge @ 13,000 rpm, 1min | ||
| + | |||
| + | |||
| + | (6) gRNA oligo ligation into plasmid | ||
| + | |||
| + | Reaction Mix | ||
| + | *3μl sense/antisense annealed oligos | ||
| + | *1μl purified vector (Kan. resistant) | ||
| + | *1μl 10x Buffer (salts, ATP, DTT, etc.) | ||
| + | *0.5μl ligase | ||
| + | *4.5μl H2O | ||
| + | —————————— | ||
| + | 10μl final vol | ||
| + | |||
| + | Master Mixes (when doing many ligations simultaneously) | ||
| + | |||
| + | (1) Master Mix #1 (mix then pipette into PCR tubes) | ||
Revision as of 13:00, 20 July 2018
(1) Oligo Rehydration
100 μmol [ ] (stock) of oligos
Ex. 100 μmol [ ] —> 27.8 nmol [ ] —> rehydrate oligo bead with 278 μl of H2O
- add H2O to hydrate oligos
- incubate 5 min at room temp
- mix
- incubate another 5 min to fully dissolve store at -20°C
(2) Sense / Antisense Oligo Annealation
TRIS Buffer
- sufficient to allow oligos to anneal
- proper pH (7.5-8)
- 10 mmol TRIS
2 μl sense oligo, 2 μl antisense oligo, 46 μl TRIS buffer (10 mmol) ————————— 50 μl final vol
- incubate samples at high temp to low temp (about -1°C / sec) - must reach 95°C
- store at 4°C
(3) Plasmid Digestion
Plasmid DR274 - digest with restriction enzyme BsaI
Always mix then centrifuge reaction components after removing from -20°
Reaction Mix (Add in parenthetical order.)
- 14.5μl H2O
- 1μl plasmid DNA (1-2 μg) (1.419 μg/μl —> just use 1 μl)
- 2μl Buffer IV (10x)
- 2μl BSA (10x)
- 10U/μl BsaI (0.5 μl for example)
——————————
20 μl final volume
(4) Gel- Plasmid Digestion
Gel Digestion
- 1.5% agarose gel
- 20 μl digestion vol + 3 μl loading dye
- run for 40 min @ 130 V
- take picture quickly! (avoid excessive UV exposure to nucleic acid)
(5) Gel- DNA Extration/Purification of Plasmid
DNA Extraction Kit (Gel Extraction) weigh eppendorf mct tube — slice out band from gel
- place in tube
- weigh again
- add volume of Buffer 1 equivalent to weight difference (gel slice weight) — incubate at 60°C for 5-10 min to let gel dissolve in extraction buffer
(should look yellow. pink/red means too basic)
- apply solution to column
- incubate for 1 min
- centrifuge @ 13,000 rpm, 1 min
- put column back to the original tube
- add 500 μl wash B (salt + EtOH)
- centrifuge 13,000 rpm 1-1.5 min
- put column back in column tube
- centrifuge @ 13,000 rpm, 2 min
- put column into new 1.5 mL Eppendorf microcentrifuge tube — add 20ul H2O to column
- incubate 1 min
- centrifuge @ 13,000 rpm, 1 min
- add 20ul H2O to column
- incubate 1 min
- centrifuge @ 13,000 rpm, 1min
(6) gRNA oligo ligation into plasmid
Reaction Mix
- 3μl sense/antisense annealed oligos
- 1μl purified vector (Kan. resistant)
- 1μl 10x Buffer (salts, ATP, DTT, etc.)
- 0.5μl ligase
- 4.5μl H2O
——————————
10μl final vol
Master Mixes (when doing many ligations simultaneously)
(1) Master Mix #1 (mix then pipette into PCR tubes)