Koeris/Notebook/2007-1-15

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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==
 
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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 !! Primer concentration [2 pmol] !! Primer concentration [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 2. 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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[[Image:2007-01-15 2pmol primer.tif|thumb|Description]]
 
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[[Image:2007-01-15 4pmol primer.tif|thumb|Description]]
 
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=Ordering=
 
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Ordered Eppendorf PCR 8-tube strips with caps; Fisher# NC9370791 @ $140
 

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