Difference between revisions of "Davidson Missouri W/colony PCR"
| (12 intermediate revisions by 2 users not shown) | |||
| Line 1: | Line 1: | ||
This procedure uses PCR to determine the size of DNA cloned into a plasmid from a single colony on a transformation plate, while reserving some of the bacteria for further growth and plasmid preparation. | This procedure uses PCR to determine the size of DNA cloned into a plasmid from a single colony on a transformation plate, while reserving some of the bacteria for further growth and plasmid preparation. | ||
| − | 1. Determine the number of colonies to be tested. Plan to conduct PCR on control plasmids with and without the insert. | + | 1. Determine the number of colonies to be tested. Plan to conduct PCR on control plasmids with and without the insert. |
| − | 2. | + | 2. Recipe for each reaction: ('''TIP: 1 uM = 1 pmol/uL''') |
| − | *1 | + | *20 pmol primer 1 (typically G00100) |
| − | * | + | *20 pmol primer 2 (typically G00101) |
| − | * | + | *10 uL 2X Promega GoTaq Hot Start Green Master ("Monster") Mix (http://www.promega.com/products/pcr/hot-start-pcr/gotaq-hot-start-polymerase/) |
| + | *Add dH2O for a '''total volume of 18 ul'''. | ||
| + | *'''If carrying out a large number of reactions, make a master mix of the water, 2X GoTaq, and primers''' | ||
| − | + | 3. Label a sterile 1.5 ml tube for each colony and aliquot 20 ul of media with the appropriate antibiotic into it. You must get some bacteria from a putative colony into both the PCR reaction and into media for growing later. Set a micripipette on 2 ul. Pick the colony off the plate and then dip it into 20 ul of media with antibiotic. Pipette up and down 3-4 times. Then use the same tip to draw up 2 ul of the media and transfer it to the PCR mixture. Pipette up and down 3-4 times. Incubate the 1.5-mL tubes at 37 C. | |
| − | + | 5. Conduct PCR according to the following thermal profile: | |
| − | + | *a. 94 C 10 minutes | |
| + | *b. 20 cycles of: 94 C (15 seconds), 46 C (15 seconds), 74 C (60 seconds for each 1000 bp of product) | ||
| − | + | 6. Run 16-20 ul on a polyacrylamide or agarose gel. The distance between the primer binding sites and your part will add bases to your product size: | |
| − | + | *Using G00100 (forward primer) adds 157 bp to parts in pSB1A2 | |
| + | *G00100 adds 588 bp to pSB1A7 parts | ||
| + | *G00100 adds 315 bp to pSB1C3 parts | ||
| − | * | + | *G00101 (reverse primer) adds 75 bp to pSB1A2 parts |
| − | * | + | *G00101 adds 302 bp to pSB1A7 parts |
| + | *G00101 adds 164 bp to pSB1C3 parts | ||
| − | + | 7. Grow desired clones from reserved bacteria for use in plasmid preps. | |
| − | |||
| − | |||
Latest revision as of 14:41, 21 January 2012
This procedure uses PCR to determine the size of DNA cloned into a plasmid from a single colony on a transformation plate, while reserving some of the bacteria for further growth and plasmid preparation.
1. Determine the number of colonies to be tested. Plan to conduct PCR on control plasmids with and without the insert.
2. Recipe for each reaction: (TIP: 1 uM = 1 pmol/uL)
- 20 pmol primer 1 (typically G00100)
- 20 pmol primer 2 (typically G00101)
- 10 uL 2X Promega GoTaq Hot Start Green Master ("Monster") Mix (http://www.promega.com/products/pcr/hot-start-pcr/gotaq-hot-start-polymerase/)
- Add dH2O for a total volume of 18 ul.
- If carrying out a large number of reactions, make a master mix of the water, 2X GoTaq, and primers
3. Label a sterile 1.5 ml tube for each colony and aliquot 20 ul of media with the appropriate antibiotic into it. You must get some bacteria from a putative colony into both the PCR reaction and into media for growing later. Set a micripipette on 2 ul. Pick the colony off the plate and then dip it into 20 ul of media with antibiotic. Pipette up and down 3-4 times. Then use the same tip to draw up 2 ul of the media and transfer it to the PCR mixture. Pipette up and down 3-4 times. Incubate the 1.5-mL tubes at 37 C.
5. Conduct PCR according to the following thermal profile:
- a. 94 C 10 minutes
- b. 20 cycles of: 94 C (15 seconds), 46 C (15 seconds), 74 C (60 seconds for each 1000 bp of product)
6. Run 16-20 ul on a polyacrylamide or agarose gel. The distance between the primer binding sites and your part will add bases to your product size:
- Using G00100 (forward primer) adds 157 bp to parts in pSB1A2
- G00100 adds 588 bp to pSB1A7 parts
- G00100 adds 315 bp to pSB1C3 parts
- G00101 (reverse primer) adds 75 bp to pSB1A2 parts
- G00101 adds 302 bp to pSB1A7 parts
- G00101 adds 164 bp to pSB1C3 parts
7. Grow desired clones from reserved bacteria for use in plasmid preps.
