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| =Pseudomonas aeruginosa mer operon cloning 3=
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| ==Preparation==
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| What can I do in order to make sure the cloning works this time?
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| *Check concentrations of the chromatin DNA: '''351 ng/ul'''
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| Remember its not pure DNA but rather cracked open cells, still yield is appropriate.
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| *Do a dilution series over two orders of magnitude of the template DNA: 3ng/ul - 300ng/ul
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| **All dilutions are to be done in at '''LEAST 100ul volume''' ddH<sub>2</sub>O
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| **Dilute stock 1:1.2: yields 300ng/ul
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| **Dilute twice 1:10: yields 30ng/ul and 3ng/ul
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| ==PCR set-up of the operon==
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| 50 ul total reaction volume
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| ===Master mix (2 pmol primers) 10X===
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| #Primers (F&R): 20 ul
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| #Taq polymerase standard reaction buffer (10X): 50 ul
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| #dNTP mix (40mM): 10 ul, i.e. final conc. 4mM/Rx
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| #Taq DNA polymerase (5U/ul): 5 ul, i.e. final conc. 0.5U/Rx
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| #ddH<sub>2</sub>O: 320 ul
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| ===Master mix (4 pmol primers) 10X===
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| #Primers (F&R): 40 ul
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| #Taq polymerase standard reaction buffer (10X): 50 ul
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| #dNTP mix (40mM): 10 ul, i.e. final conc. 4mM/Rx
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| #Taq DNA polymerase (5U/ul): 5 ul, i.e. final conc. 0.5U/Rx
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| #ddH<sub>2</sub>O: 300 ul
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| ===Pipetting scheme===
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| Labels in the respective field codes
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| {| border="1"
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| ! Template DNA concentration !! Primerconcentration [2 pmol] !! Primerconcentration [4 pmol]
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| |-
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| ! 3 ng
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| | A1
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| | B2
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| |-
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| ! 6 ng
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| | A2
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| | B2
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| |-
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| ! 9 ng
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| | A3
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| | B3
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| |-
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| ! 30 ng
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| | A4
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| | B4
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| |-
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| ! 60 ng
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| | A5
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| | B5
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| |-
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| ! 90 ng
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| | A6
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| | B6
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| |-
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| ! 150 ng
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| | A7
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| | B7
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| |-
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| ! 300 ng
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| | A8
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| | B8
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| |}
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| ===Thermal profile===
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| #92 deg C - 5min
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| #92 deg C - 30s
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| #50 deg C - 45s
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| #72 deg C - 120s
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| #Cycle back to 92 deg C 29X
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| #72 deg C - 10min
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| #4 deg C - indefinite
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| ==Gel image==
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| Two 14-comb gels @ 50ml
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| Loading volume: 20ul of PCR Rx = 40% product
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| 1% agarose TAE gel, ran @ 100V for 60min
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