Assembly Related Protocols
From 2008.igem.org
Contents |
Combining basic part with assemblies
- PCR
- =25ul
- Purfication using gel purification
- cutting out gel band
- cut out band (if correct size)
- dissolve gel in 3 vol of ADB @ 55 celcius, for 5-10 min.
- the "Lazy way"
- take 5ul of PCR product
- add a little bit of loading dye and place in gel well
- check if band size is correct, then zymo remaining 20ul.
- cutting out gel band
- Digest
- basic part
- EcoRI/BamHI/DpnI
- Digest with 50 ul scale (digest for 1 hour)
- Assembly + vector
- take 4ul part
- 4ul water
- 1 ul NEB2
- 0.5 ul EcoRI
- 0.5 ul BglII
- basic part
- ZYMO BOTH simultaneously
- part: elute with 10ul water
- AVector: elute with 6ul water
- Ligation (sit for 30 min)
- 1ul part
- 1ul AVector
- 1ul Ligase buffer
- 0.5ul Ligase
- Transform
Newest EcoRI/BamHI Transfer method (7/31/08)
Digestion:
- 0.5ul plasmid in entry vector
- 0.5ul EcoRI
- 0.5ul BamHI
- 1ul NEB2 + ATP buffer
- 7.5ul of water
- incubate the digestion for 1hr. in the thermocycler, and 20 min for heat kill.
No purification step.
Ligation:
- 1ul of dilute assembly vector (or 0.5ul if undiluted)
- 0.5ul ligase
Transform. (same as usual)
Transfers from Miniprepped DNA
(already in correct vectors - transfers into L/R cells)
In 0.5 ml tube, combine:
- 1ul plasmid dna
- 30ul of cells (L/R) + 30 KCM (no water)
Take the reaction, and:
- sit in ice for 10 min
- heat shock for 90 sec
- sit another 2 min ice
Then, incubate your tubes for 5 minutes (No rescuing is necessary)
Next, prepare plates (double antibiotics) and tubes with media (LB + antibiotics)
- Take 15ul of rxn. Spot it onto a plate (no spreading required)
- Take the remaining 15ul into a pipette tip. Drop the entire tip into your tube of media.
- Let plates and tubes sit overnight.
- Miniprep the next day.
1-2-3 Assembly
Set up the digestion:
- 1uL lefty plasmid(methylated)
- 1uL righty plasmid (methylated)
- 1uL NEB2+ATP
- 0.3 uL XhoI
- 0.3 uL BglII
- 0.3 uL BamHI
- 6.1uL Water
Heat kill at 65 for 20 min
Add 0.3uL Ligase
Transform, rescue, plate on dual antibiotic plates
Dealing with Co-transformation (8/4/08)
Method 1: Pick more white colonies...
Method 2: Seperating the 2 plasmids:
- Take miniprepped, cotransformed dna sample, and dilute 100x
- Take 1ul of diluted DNA to transform
- Use selected cell strain
- rescue 15 min in incubator
- Grow on double-antibiotic plate overnight
- pick colonies (maybe 2)
- streak on double antibiotic plate, and amp or spec to check for co-transformation
- simultaneously grow in media with corresponding double antibiotics
- miniprep the next day
Method 3: "Removing" the cotransformant plasmid
- pick colonies from the restreaked plate (the ones that are for sure cotransformed
- grow in double antibiotic media overnight
- dip toothpick into culture from night before, and restreak on double antibiotic plate
- the next day, pick colonies and screen for cotransformation