Reverse Parts with PCR
From 2006.igem.org
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Restriction digest as follows: | Restriction digest as follows: | ||
| - | + | # jfak | |
| + | # fkja | ||
these two things in separate tubes with both XbaI and SpeI | these two things in separate tubes with both XbaI and SpeI | ||
Revision as of 11:50, 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:
- jfak
- fkja
these two things in separate tubes with both XbaI and SpeI
1. Band from PCR 2. plasmid I13522 (pTet-GFP, glows green with UV)
Design and set up ligations
1. 20 ng pTet-GFP X/S only 2. 20 ng pTet-GFP X/S + 10 ng pARA X/S
Transform JM109 cells, along with the hix ligations (take out one tube of 200 ul cells and divide it among the transformations, probably 200/6 = 33 ul each)
