Reverse Parts with PCR
From 2006.igem.org
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Restriction digest as follows: | Restriction digest as follows: | ||
| + | 1. 20 ul eluted PCR product | ||
| + | 2. 16 ul dH2O | ||
| + | 3. 2 ul NEB2 10X buffer | ||
| + | 4. 1 ul XbaI | ||
| + | 5. 1 ul SpeI | ||
| + | Heat inactivate the restriction enzymes by putting the tubes at 65C for 20 minutes | ||
| - | + | Set up ligations | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
1. 20 ng pTet-GFP X/S only | 1. 20 ng pTet-GFP X/S only | ||
| - | 2. 20 ng pTet-GFP X/S + | + | 2. 20 ng pTet-GFP X/S + 5 ul X/S digest from above |
| - | Transform JM109 cells | + | Transform JM109 cells (take out one tube of 200 ul cells and divide it among the transformations) |
Latest revision as of 11:55, 21 June 2006
Dissolving PCR primers
1. spin down tubes with dry DNA 2. calculate volume dH2O to give 200 pmol/ul, label as "10X" 3. dilute 10X to make 1X
Conduct PCR (30 cycles, use Tm to determine annealing temp)
1. 1 ul Forward primer 2. 1 ul Reverse primer 3. 2 ul 10X with no Mg 4. 2 ul 25 mM MgCl2 5. 12 ul dH2O 6. 1 ul Taq 7. 1 ul DNA template
DNA template: Add 15 ul dH2O to appropriate well on plate DNA-1 or DNA-2. Take 1 ul of this and add it to 20 ul water and label as "1/20" then part number. Use 1 ul of this in PCR.
After PCR, add 5 ul 5X loading buffer and run on 7% PA gel (product should be 150 bp)
If this works, scale up the PCR to 5 x 20 ul, run new PA gel, and purify band. Elute in 2 x 40 ul.
Restriction digest as follows:
1. 20 ul eluted PCR product 2. 16 ul dH2O 3. 2 ul NEB2 10X buffer 4. 1 ul XbaI 5. 1 ul SpeI
Heat inactivate the restriction enzymes by putting the tubes at 65C for 20 minutes
Set up ligations
1. 20 ng pTet-GFP X/S only 2. 20 ng pTet-GFP X/S + 5 ul X/S digest from above
Transform JM109 cells (take out one tube of 200 ul cells and divide it among the transformations)
