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data_overview:steven_weber_s_notes_on_lab_prep

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data_overview:steven_weber_s_notes_on_lab_prep [2015/04/03 22:08]
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data_overview:steven_weber_s_notes_on_lab_prep [2015/04/03 22:10]
ed
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 3. Blunt End Repair with 50ul of sample plus the following: 3. Blunt End Repair with 50ul of sample plus the following:
  
-* 7.12ul ddH2O +  ​* 7.12ul ddH2O 
-* 7ul Buffer Tango (10x) +  * 7ul Buffer Tango (10x) 
-* 0.28ul dNTPs (25mM each) +  * 0.28ul dNTPs (25mM each) 
-* 0.7ul  ATP (100mM) +  * 0.7ul  ATP (100mM) 
-* 3.5ul PNK (10U/ul) +  * 3.5ul PNK (10U/ul) 
-* 1.4ul T4 DNA Polymerase (5U/ul)+  * 1.4ul T4 DNA Polymerase (5U/ul)
  
 Incubated at 25°C for 15 minutes then at 12°C for 5 minutes Incubated at 25°C for 15 minutes then at 12°C for 5 minutes
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 4. Adapter Ligation with 20ul sample plus the following: 4. Adapter Ligation with 20ul sample plus the following:
  
-* 10ul ddH2O +  ​* 10ul ddH2O 
-* 4ul T4 Ligase Buffer + 10mM ATP (10x) +  * 4ul T4 Ligase Buffer + 10mM ATP (10x) 
-* 4ul PEG-4000 (50%) +  * 4ul PEG-4000 (50%) 
-* 1ul P5+P7 adapters (100uM each) +  * 1ul P5+P7 adapters (100uM each) 
-* 1ul T4 DNA Ligase (5U/ul)+  * 1ul T4 DNA Ligase (5U/ul)
  
 Incubated at 22°C for 30 minutes Incubated at 22°C for 30 minutes
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 5. Adapter fill-in with 20ul sample plus the following: 5. Adapter fill-in with 20ul sample plus the following:
  
-* 14.1ul ddH2O +  ​* 14.1ul ddH2O 
-* 4ul ThermoPol Buffer (10x) +  * 4ul ThermoPol Buffer (10x) 
-* 0.4ul dNTPs (25uM each) +  * 0.4ul dNTPs (25uM each) 
-* 1.5ul Bst DNA Polymerase (8U/ul)+  * 1.5ul Bst DNA Polymerase (8U/ul)
  
 Incubated at 37°C for 20 minutes Incubated at 37°C for 20 minutes
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 Thermal Cycler Parameters Thermal Cycler Parameters
  
-* 25ul KAPA 2x Polymerase mix +  ​* 25ul KAPA 2x Polymerase mix 
-* 2.5ul sample +  * 2.5ul sample 
-* 1ul Primer IS4 (10uM) +  * 1ul Primer IS4 (10uM) 
-* 1ul indexing primer (10uM) +  * 1ul indexing primer (10uM) 
-* 20.5ul ddH2O+  * 20.5ul ddH2O
  
 1. 98°C for 3 minutes 1. 98°C for 3 minutes
data_overview/steven_weber_s_notes_on_lab_prep.txt · Last modified: 2015/07/28 05:53 by ceisenhart