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