Matt Gethers/CRI, Thailand/Labwork/PCRs/Screening for Presence and Directionality of HmgA Downstream Fragment in pUC18
Screening for Presence and Directionality of HmgA Downstream Fragment in pUC18
Rxn Mix
| Reagent | Volume/Amount |
| DNA Template | 1.0 μl from Colony Suspension |
| Primer M13 For | 1.2 μl 1 μM Stock |
| Primer 2724 | 1.2 μl 1 μM Stock |
| 2x PCR Master | 7.5 μl |
| H20 | 4.1 μl |
| Total | 15 μl |
| Reagent | Volume/Amount |
| Paeru Genomic DNA Template | 0.5 μl from So Pa Pan stock |
| Taq | 0.5 μl |
| Primer M13 For | 4 μl 1 μM Stock |
| Primer 2724 | 4 μl 1 μM Stock |
| dNTPs | 1 μl 10 mM Stock |
| DMSO | 2.5 μl |
| Buffer | 5 μl |
| Mg2+ | 2.5 μl |
| dH20 | 30 μl |
| Total | 50 μl |
Rxn Conditions
Annealing Temperature: 55oC (1 degree below 2724 annealing temp)
Extension Time: 2:15 minutes (2 minutes/Kb)
Cycle
| Step | Temperature | Duration | Notes |
| Initial Denaturation | 95oC | 3 minutes | |
| Cyclic Denaturation | 94oC | 40 Seconds | |
| Cyclic Annealing | 55oC | 40 Seconds | Unsure of best temp |
| Cyclic Extension | 72oC | 2:15 minutes | |
| Repeat Cyclic Steps 35x | |||
| Final Extension | 72oC | 10 minutes |
Cycle (Taq)
| Step | Temperature | Duration | Notes |
| Initial Denaturation | 95oC | 2 minutes | |
| Repeat Cyclic Steps 35x | |||
| Cyclic Denaturation | 94oC | 30 Seconds | |
| Cyclic Annealing | 55oC | 30 Seconds | Unsure of best temp |
| Cyclic Extension | 72oC | 1 minute | |
| Repeat Cyclic Steps 35x | |||
| Final Extension | 72oC | 10 minutes |
Run Notes
6.25.08
Run 1:
I selected 6 colonies from the plates I streaked out of what appeared to be white colonies. I inoculated these in 50 μl of H20 and made up a PCR using supermix (7.5 μl supermix, 1.2 μl M13_for, 1.2 μl BT2724, 1 μl template, 4.1 μl H20). I used 1 μl of suspension per reaction. Ran with annealing temp of 55 degrees and extension time of 2:15.
Run 2:
The supermix just isn't working for me, so I'm using a Taq PCR kit from Finnzyme instead. Made a master mix of water, magnesium, buffer, DMSO, dNTPs, and Taq, split in two, then added primers M13 for and BT2724 to one aliquot and primers BT2736 an dBT2737 to the other aliquot. Then I aliquotted 8.2 μl to each of 12 tubes, 6 for pUC18/HmgA Downstream colonies, 5 for pET-11a/HmgR, and 1 for a positive control using Genomic DNA (still using BT2736/37). I modified the protocol a bit - I made 10 μl reactions and I used 1 μl of template for each 10 μl reaction (except for the positive control in which I used 0.5 μl temp and 0.5μl water). So rather than using 5.9 μl water per reaction, I used 5.1. I'm running with an annealing temp of 55 degrees and an extension time of 1:15 minutes (taq requires shorter time than Pfu and I'm running this PCR along side the HmgA Downstream fragment PCR, hence the slightly longer time). See gel results here (6.26.08 is generated from the Taq rather than supermix)
