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Matt Gethers/CRI, Thailand/Labwork/PCRs/Screening for Presence and Directionality of HmgA Downstream Fragment in pUC18

Contents

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)