Daniel:Notebook/ComboLock/2017-3-22: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
>Djacobse
 
(11 intermediate revisions by the same user not shown)
Line 81: Line 81:
<li>Add 20 uL nfH2O</li></ol>
<li>Add 20 uL nfH2O</li></ol>
<li>TBE gel</li>
<li>TBE gel</li>
<li>Mix uL TBE and uL 6X dye</li>
<li>Mix 64 uL TBE and 16 uL 6X dye</li>
<li>Aliquot 10 uL on to parafilm per sample/ladder</li>
<li>Aliquot 10 uL on to parafilm per sample/ladder</li>
<li>Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot</li>
<li>Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot</li>
Line 92: Line 92:
<li>Rinse and image in gel doc</li>
<li>Rinse and image in gel doc</li>
</ol>
</ol>
[[Image:2017-03-22-CPProduction.png|500px]]
==Protocol Part 2==
That was unexpected. From what I'm seeing on [http://www.bioinformatics.nl/molbi/SimpleCloningLab/electrophoresis.htm the internet], this might be a thing. Circular DNA looks to appear much higher on the gel than it should. I can try and Bglii digest to be sure.
<ol start="3">
<li>Bglii digest</li>
<ol type="A">
<li>Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest</li>
<li>Make the following reactions; '''Do not add Bglii yet'''</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center"
| width="220" height="30" | Reagent
| width="105" | Single Rxn Vol
|- style="font-size:12pt"
| height="15"  valign="bottom" | Ligation Reaction Product
| align="center" align="center" valign="bottom" | 4
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Bglii Cutter Primer (10 uM)
| align="center" align="center" valign="bottom" | 5
|- style="font-size:12pt"
| height="15"  valign="bottom" | 10X Buffer 3.1
| align="center" align="center" valign="bottom" | 2
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Bglii
| align="center" align="center" valign="bottom" | 1
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 8
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Total
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20
|}
<li>Use the following thermocycler program</li>
<ol type="a">
<li>5 min 95C</li>
<li>Ramp to 50C at 0.2C/s</li>
<li>10 min 50C</li>
<li>1 hr 37C; '''When this cycle starts add 1 uL Bglii'''</li>
<li>Heat kill with 65C for 20 min</li></ol>
</ol>
<li>Qiaquick Column Purification (Circular product reaction from part 1)</li>
<ol type="A">
<li>Add 200 uL PB to sample</li>
<li>Centrifuge at 13000rpm for 1 minute</li>
<li>Dump flow through and add 700 uL PE buffer</li>
<li>Centrifuge at 13000rpm for 1 minute</li>
<li>Dump flow through and add 700 uL PE buffer</li>
<li>Centrifuge at 13000rpm for 1 minute</li>
<li>Dump flow through and centrifuge empty for 2 min at 13000 rpm</li>
<li>Add 50 uL nfH2O and centrifuge for 2 min at 13000 rpm</li></ol>
<li>TBE Gel II</li>
<ol type="A">
<li>Mix 80 uL TBE and 20 uL 6X dye</li>
<li>Aliquot 10 uL on to parafilm per sample/ladder</li>
<li>Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot</li>
[[Image:GelLanes2-20170322-Circ.png|400px|Gel lanes]]
<li>Add 10 uL mix to gel lanes</li>
<li>Run gel for 24 minutes at 230V</li>
<li>Stain with 2 uL SYBR gold for 3 minutes</li>
<li>Rinse and image in gel doc</li>
</ol></ol>
[[Image:2017-03-22-CPProduction-2.png|500px]]
[[Category:ComboLock]] [[Category:20170322]]

Latest revision as of 22:05, 23 March 2017

Circular Product Production[edit]

Back to Calendar

The goal of this will be to produce a large amount of circular product for use in testing.

Protocol[edit]

  1. Ligation
    1. Make reaction mixture according to table; Do not add ligase yet
    2. Reagent Stock Conc Final Conc./Amount uL added
      T4 Ligase Buffer 5X 1X 4
      PosCon Circ Latch Oligo 100 uM 200 pmol 1
      Padlock 0601 oligo 100 uM 200 pmol 1
      C4 Positive Control oligo 100 uM 200 pmol 1
      C2 positive Control oligo 100 uM 200 pmol 1
      T4 DNA Ligase 5 U/uL 5 U 1
      nfH2O NA NA 11
      Total     20
    3. Use following thermocycler program
      1. Heat mixture to 95C for 5 min
      2. Lower temp by 0.2C per second to 55C
      3. Hold mixture at 55C for 15 minutes
      4. Lower temp to 20C by reducing 0.2C per second
      5. Hold at 20C
    4. Add 1 uL T4 DNA ligase
    5. Incubate at 20C for 30 minutes
    6. Heat kill enzyme by incubating at 65C for 15 minutes
    7. Add 20 uL nfH2O
  2. TBE gel
  3. Mix 64 uL TBE and 16 uL 6X dye
  4. Aliquot 10 uL on to parafilm per sample/ladder
  5. Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot
  6. Gel lanes
  7. Add 10 uL mix to gel lanes
  8. Run gel for 24 minutes at 230V
  9. Stain with 2 uL SYBR gold for 3 minutes
  10. Rinse and image in gel doc

File:2017-03-22-CPProduction.png

Protocol Part 2[edit]

That was unexpected. From what I'm seeing on the internet, this might be a thing. Circular DNA looks to appear much higher on the gel than it should. I can try and Bglii digest to be sure.

  1. Bglii digest
    1. Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
    2. Make the following reactions; Do not add Bglii yet
    3. Reagent Single Rxn Vol
      Ligation Reaction Product 4
      Bglii Cutter Primer (10 uM) 5
      10X Buffer 3.1 2
      Bglii 1
      nfH2O 8
      Total 20
    4. Use the following thermocycler program
      1. 5 min 95C
      2. Ramp to 50C at 0.2C/s
      3. 10 min 50C
      4. 1 hr 37C; When this cycle starts add 1 uL Bglii
      5. Heat kill with 65C for 20 min
  2. Qiaquick Column Purification (Circular product reaction from part 1)
    1. Add 200 uL PB to sample
    2. Centrifuge at 13000rpm for 1 minute
    3. Dump flow through and add 700 uL PE buffer
    4. Centrifuge at 13000rpm for 1 minute
    5. Dump flow through and add 700 uL PE buffer
    6. Centrifuge at 13000rpm for 1 minute
    7. Dump flow through and centrifuge empty for 2 min at 13000 rpm
    8. Add 50 uL nfH2O and centrifuge for 2 min at 13000 rpm
  3. TBE Gel II
    1. Mix 80 uL TBE and 20 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample/ladder
    3. Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot
    4. Gel lanes
    5. Add 10 uL mix to gel lanes
    6. Run gel for 24 minutes at 230V
    7. Stain with 2 uL SYBR gold for 3 minutes
    8. Rinse and image in gel doc

File:2017-03-22-CPProduction-2.png