Koeris/Notebook/2007-1-15: Difference between revisions

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**Dilute twice 1:10: yields 30ng/ul and 3ng/ul
**Dilute twice 1:10: yields 30ng/ul and 3ng/ul
==PCR set-up of the operon==
==PCR set-up of the operon==
#Master mix
50 ul total reaction volume
##Template DNA equiv.  
===Master mix (2 pmol primers) 10X===
##ddH<sub>2</sub>O to 50uL
#Primers (F&R): 20 ul
#Thermal profile
#Taq polymerase standard reaction buffer (10X): 50 ul
##92 deg C - 5min
#dNTP mix (40mM): 10 ul, i.e. final conc. 4mM/Rx
##92 deg C - 30s
#Taq DNA polymerase (5U/ul): 5 ul, i.e. final conc. 0.5U/Rx
##50 deg C - 45s
#ddH<sub>2</sub>O:
##72 deg C - 120s
 
##4 deg C - indefinite
===Pipetting scheme===
===Pipetting scheme===
Labels in the respective field codes
Labels in the respective field codes
Line 54: Line 53:
| B8
| B8
|}
|}
 
===Thermal profile===
#92 deg C - 5min
#92 deg C - 30s
#50 deg C - 45s
#72 deg C - 120s
#4 deg C - indefinite
==Gel image==
==Gel image==
1% agarose TAE gel, ran @ 100V for 60min
1% agarose TAE gel, ran @ 100V for 60min

Revision as of 09:33, 15 January 2007

Pseudomonas aeruginosa mer operon cloning 3

Preparation

What can I do in order to make sure the cloning works this time?

  • Check concentrations of the chromatin DNA: 351 ng/ul
  • Do a dilution series over two orders of magnitude of the template DNA: 3ng/ul - 300ng/ul
    • All dilutions are to be done in at LEAST 100ul volume ddH2O
    • Dilute stock 1:1.2: yields 300ng/ul
    • Dilute twice 1:10: yields 30ng/ul and 3ng/ul

PCR set-up of the operon

50 ul total reaction volume

Master mix (2 pmol primers) 10X

  1. Primers (F&R): 20 ul
  2. Taq polymerase standard reaction buffer (10X): 50 ul
  3. dNTP mix (40mM): 10 ul, i.e. final conc. 4mM/Rx
  4. Taq DNA polymerase (5U/ul): 5 ul, i.e. final conc. 0.5U/Rx
  5. ddH2O:

Pipetting scheme

Labels in the respective field codes

Template DNA concentration Primerconcentration [2 pmol] Primerconcentration [4 pmol]
3 ng A1 B2
6 ng A2 B2
9 ng A3 B3
30 ng A4 B4
60 ng A5 B5
90 ng A6 B6
150 ng A7 B7
300 ng A8 B8

Thermal profile

  1. 92 deg C - 5min
  2. 92 deg C - 30s
  3. 50 deg C - 45s
  4. 72 deg C - 120s
  5. 4 deg C - indefinite

Gel image

1% agarose TAE gel, ran @ 100V for 60min

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