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] ed created |
data_overview:steven_weber_s_notes_on_lab_prep [2015/07/28 05:53] (current) ceisenhart ↷ Page moved from steven_weber_s_notes_on_lab_prep to data_overview:steven_weber_s_notes_on_lab_prep |
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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 | ||
| Line 116: | Line 116: | ||
| 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 | ||
| Line 128: | Line 128: | ||
| 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 | ||
| Line 155: | Line 155: | ||
| 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.1428098932.txt.gz · Last modified: 2015/04/03 22:08 by ed
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