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

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Contents

Screening for Presence and Directionality of HmgA Downstream Fragment in pUC18

Rxn Mix

Reagent Volume/Amount
DNA Template1.0 μl from Colony Suspension
Primer M13 For1.2 μl 1 μM Stock
Primer 27241.2 μl 1 μM Stock
2x PCR Master7.5 μl
H204.1 μl
Total15 μl
Reagent Volume/Amount
Paeru Genomic DNA Template0.5 μl from So Pa Pan stock
Taq0.5 μl
Primer M13 For4 μl 1 μM Stock
Primer 27244 μl 1 μM Stock
dNTPs1 μl 10 mM Stock
DMSO 2.5 μl
Buffer 5 μl
Mg2+ 2.5 μl
dH2030 μl
Total50 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 Denaturation95oC3 minutes
Cyclic Denaturation94oC40 Seconds
Cyclic Annealing55oC40 SecondsUnsure of best temp
Cyclic Extension72oC2:15 minutes
Repeat Cyclic Steps 35x
Final Extension72oC10 minutes

Cycle (Taq)

Step Temperature Duration Notes
Initial Denaturation95oC2 minutes
Repeat Cyclic Steps 35x
Cyclic Denaturation94oC30 Seconds
Cyclic Annealing55oC30 SecondsUnsure of best temp
Cyclic Extension72oC1 minute
Repeat Cyclic Steps 35x
Final Extension72oC10 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)

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