20.109(S13):DNA cloning (Day4)

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===Part 3: Transform ligated product into cloning strain===
===Part 3: Transform ligated product into cloning strain===
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===Set up microsporidia PCR on this day?===
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<font color=red> need to do microsporidia PCR on separate day because the Ta etc. is different --> need to find notes on where I planned to put that. possibly Day 2? primers should have arrived by then. or maybe wait until Day 4? don't want to have them balancing lab steps too soon.</font color>
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#Begin by carefully labeling each PCR tube that you will use with the date, sample name, and a unique symbol and/or color for quick identification. Filling in the cap tab with your team color will usually suffice. Pre-chill the tubes on a cold block.
 +
#You and your partner can now prepare and share a so-called "master mix," which contains every PCR ingredient except the template and the polymerase. Prepare enough for the number of reactions you need to run, plus an additional 10%. Feel free to use the table below for your calculations.
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#*When the master mix is not in use, keep it on ice.
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#Combine 5 &mu;L of template, 45 &mu;L of master mix, and 1 &mu;L of ''PfuUltra'' polymerase in a PCR tube. When everyone's reactions are ready, they will undergo the cycling conditions listed below.
 +
#*Add the master mix first, because the template alone may freeze. Then add template and polymerase, and finally (gently!) mix the reaction with a larger pipet.
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<center>
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{| border="1"
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! Reagent
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! Amount for 1 reaction (&mu;L)
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! Amount for ? reactions + 10%
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|-
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| ''PfuUltra'' buffer (10X stock)
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| 5
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|
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|-
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| Primer mix [TO WHAT EXTENT PREPARED FOR THEM?]
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| 5 of ''each'' (Fx and Rx)
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|
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|-
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| dNTPs
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| 1
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|
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|-
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| 10% BSA (100X stock)
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| 0.5
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|
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|-
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| Water
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| 28.5
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|
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|-
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| DNA template
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| 5
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| N/A
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|-
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|}
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</center>
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<center>
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{| border="1"
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! Segment
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! Cycles
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! Temperature (&deg; C)
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! Time
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|-
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| 1
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| 1
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| 95
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| 3 min
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|-
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| 2-4
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| 40
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| 95
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| 1 min
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|-
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|
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|
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| 58
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| 1.5 min
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|-
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|
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|
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| 72
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| 2 min
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|-
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| 5
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| 1
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| 72
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| 10 min
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|-
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| 6
 +
| 1
 +
| 4
 +
| indefinite
 +
|}
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</center>
==For next time==
==For next time==

Revision as of 13:53, 10 January 2013

20.109(S13): Laboratory Fundamentals of Biological Engineering

Home        Schedule Spring 2013        Assignments       
DNA Engineering        Protein Engineering        Cell Engineering              

Contents

Introduction

background on this cloning system -- vector arms and strain

transformation

Protocols

Part 1: Purify 16S PCR band from gel

Part 2: Ligate purified product to vector arms

Part 3: Transform ligated product into cloning strain

Set up microsporidia PCR on this day?

need to do microsporidia PCR on separate day because the Ta etc. is different --> need to find notes on where I planned to put that. possibly Day 2? primers should have arrived by then. or maybe wait until Day 4? don't want to have them balancing lab steps too soon.

  1. Begin by carefully labeling each PCR tube that you will use with the date, sample name, and a unique symbol and/or color for quick identification. Filling in the cap tab with your team color will usually suffice. Pre-chill the tubes on a cold block.
  2. You and your partner can now prepare and share a so-called "master mix," which contains every PCR ingredient except the template and the polymerase. Prepare enough for the number of reactions you need to run, plus an additional 10%. Feel free to use the table below for your calculations.
    • When the master mix is not in use, keep it on ice.
  3. Combine 5 μL of template, 45 μL of master mix, and 1 μL of PfuUltra polymerase in a PCR tube. When everyone's reactions are ready, they will undergo the cycling conditions listed below.
    • Add the master mix first, because the template alone may freeze. Then add template and polymerase, and finally (gently!) mix the reaction with a larger pipet.
Reagent Amount for 1 reaction (μL) Amount for ? reactions + 10%
PfuUltra buffer (10X stock) 5
Primer mix [TO WHAT EXTENT PREPARED FOR THEM?] 5 of each (Fx and Rx)
dNTPs 1
10% BSA (100X stock) 0.5
Water 28.5
DNA template 5 N/A
Segment Cycles Temperature (° C) Time
1 1 95 3 min
2-4 40 95 1 min
58 1.5 min
72 2 min
5 1 72 10 min
6 1 4 indefinite

For next time

Reagent list

write something here or not accessible to edit
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