Making Reverse pARA
From 2006.igem.org
(Difference between revisions)
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# 150 bp pAra band from above | # 150 bp pAra band from above | ||
# plasmid I13522 (pTet-GFP, glows green with UV) | # plasmid I13522 (pTet-GFP, glows green with UV) | ||
+ | |||
+ | Restriction digest as follows: | ||
+ | |||
+ | 1. 20 ul eluted PCR product or pTet-GFP | ||
+ | 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 | ||
Design and set up ligations | Design and set up ligations | ||
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# 20 ng pTet-GFP X/S + 10 ng pARA X/S | # 20 ng pTet-GFP X/S + 10 ng pARA X/S | ||
- | Transform JM109 cells | + | 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) |
Latest revision as of 11:58, 21 June 2006
PCR primers arrive Tuesday
- spin down tubes with dry DNA
- calculate volume dH2O to give 200 pmol/ul, label as "10X"
- dilute 10X to make 1X
Conduct PCR (30 cycles, use Tm to determine annealing temp)
- 1 ul Forward primer
- 1 ul Reverse primer
- 2 ul 10X with no Mg
- 2 ul 25 mM MgCl2
- 12 ul dH2O
- 1 ul Taq
- 1 ul plasmid with pARA (labeled I13453, in right freezer)
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.
Digest these two things in separate tubes with both XbaI and SpeI
- 150 bp pAra band from above
- plasmid I13522 (pTet-GFP, glows green with UV)
Restriction digest as follows:
1. 20 ul eluted PCR product or pTet-GFP 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
Design and set up ligations
- 20 ng pTet-GFP X/S only
- 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)