Materials
· Primers @ 20pmol/ul concentration
· Taq
· rxn buffer
· 25mM MgCl2 (optional -- do not optimize Mg conc. when using Klentaq; when using Promega Taq, optimize from 1mM-3.5mM in .5mM increments)
· 2mM dNTPs
· ddH2O (filter sterilized, non-autoclaved)
· Sterile pipette tips (aerosol tips are not necessary -- it is recommended to use tips that have not been autoclaved, but I have also not found this necessary.)
· Sterile PCR tubes (when using non PCR .5ml eppies, the lids can pop open -- the lids can also pop open when using PCR tubes so be sure to close them tightly)
Prepare Reactions on Ice
The exception to this is when using Klentaq -- there is an antibody which renders the enzyme inactive until it is heated to 95 degrees so it will not catalyze any annealing prior reaching 95 degrees.
1. Streak the strain of interest for single colonies about 15 hours before using for E. coli and 24-48 hours prior to using for C. crescentus. Growth is strain dependent, but try to select an average colony for amplification.
2. Determine melting temperature for primers. Sometimes starting with a lower annealing temperature (45 degrees) and then gradually increasing (2 degrees per trial is best for optimizing this aspect of the reaction).
3. Determine extension time (recommended 1 min. per 1 kb, but when looking for larger fragments, more time can be better.
4. Determine extension temperature (read the Taq product info -- use 68 degrees for Klentaq and 72 degrees for Promega despite the recommended 74 degrees).
5. Make reaction cocktails (follow PCR sample log as a guide -- however, notes on here have been made for using Klentaq to obtain specific cgtA products -- please alter dNTPs to 5�l/50¨�l reaction when using Promega Taq -- also be sure to adjust times and temperatures under the thermocylcer entry) -- another note about reaction cocktails -- when optimizing Mg concentration then add as many things to the cocktail as possible and supplement the reaction with appropriate amounts of Mg and ddH2O, and it is a good idea to make a larger volume cocktail than believed necessary because of pipetting errors.
6. Add reaction mix and supplements to PCR tube -- spin down volume if necessary
7. Add single colony to reaction mix by picking the colony with either a sterile loop or pipette tip (do not use wood) and twirling this in the liquid in the tube.
8. DO NOT spin the tube down after adding the single colony -- this might pellet the cells that were added.
9. Begin the PCR program -- when the block reaches 95 degrees, press pause, add the reactions, and then continue the cycle -- if you add the reactions prior to 95 degrees and you are not using Klentaq then you can get primer/dimer formation which will inhibit your reactions.
· Primers @ 20pmol/ul concentration
· Taq
· rxn buffer
· 25mM MgCl2 (optional -- do not optimize Mg conc. when using Klentaq; when using Promega Taq, optimize from 1mM-3.5mM in .5mM increments)
· 2mM dNTPs
· ddH2O (filter sterilized, non-autoclaved)
· Sterile pipette tips (aerosol tips are not necessary -- it is recommended to use tips that have not been autoclaved, but I have also not found this necessary.)
· Sterile PCR tubes (when using non PCR .5ml eppies, the lids can pop open -- the lids can also pop open when using PCR tubes so be sure to close them tightly)
Prepare Reactions on Ice
The exception to this is when using Klentaq -- there is an antibody which renders the enzyme inactive until it is heated to 95 degrees so it will not catalyze any annealing prior reaching 95 degrees.
1. Streak the strain of interest for single colonies about 15 hours before using for E. coli and 24-48 hours prior to using for C. crescentus. Growth is strain dependent, but try to select an average colony for amplification.
2. Determine melting temperature for primers. Sometimes starting with a lower annealing temperature (45 degrees) and then gradually increasing (2 degrees per trial is best for optimizing this aspect of the reaction).
3. Determine extension time (recommended 1 min. per 1 kb, but when looking for larger fragments, more time can be better.
4. Determine extension temperature (read the Taq product info -- use 68 degrees for Klentaq and 72 degrees for Promega despite the recommended 74 degrees).
5. Make reaction cocktails (follow PCR sample log as a guide -- however, notes on here have been made for using Klentaq to obtain specific cgtA products -- please alter dNTPs to 5�l/50¨�l reaction when using Promega Taq -- also be sure to adjust times and temperatures under the thermocylcer entry) -- another note about reaction cocktails -- when optimizing Mg concentration then add as many things to the cocktail as possible and supplement the reaction with appropriate amounts of Mg and ddH2O, and it is a good idea to make a larger volume cocktail than believed necessary because of pipetting errors.
6. Add reaction mix and supplements to PCR tube -- spin down volume if necessary
7. Add single colony to reaction mix by picking the colony with either a sterile loop or pipette tip (do not use wood) and twirling this in the liquid in the tube.
8. DO NOT spin the tube down after adding the single colony -- this might pellet the cells that were added.
9. Begin the PCR program -- when the block reaches 95 degrees, press pause, add the reactions, and then continue the cycle -- if you add the reactions prior to 95 degrees and you are not using Klentaq then you can get primer/dimer formation which will inhibit your reactions.
