Daniel:Notebook/ComboLock/2017-3-25: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
>Djacobse
 
(9 intermediate revisions by the same user not shown)
Line 22: Line 22:
</ol></ol>
</ol></ol>


==RCA Protocol==
[[Image:2017-03-25-LinearProductProduction.png|400px]]


<ol>
Still too much circular product present. I'll try one more time to make it linear.
<li>Rolling Circle Amplification</li>
 
==Linear Product Production==
 
This time we'll start with the circular product and chop it.
 
<ol start="3">
<li>Bglii digest</li>
<ol type="A">
<ol type="A">
<li>Prepare master mix according to table below</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>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center"
| width="200" height="30" | Reagent
| width="220" height="30" | Reagent
| width="85" | Single Rxn (uL)
| width="105" | Single Rxn Vol
| width="85" | Master Mix X+ (6.2X)
| width="85" | Master Mix X- (6.2X)
| width="85" | Master Mix Y+ (3.2X)
| width="85" | Master Mix Y- (3.2X)
| width="85" | Master Mix Z+ (3.2X)
| width="85" | Master Mix Z- (3.2X)
| width="85" | Master Mix P+ (3.2X)
| width="85" | Master Mix P- (3.2X)
| width="85" | Master Mix Q+ (3.2X)
| width="85" | Master Mix Q- (3.2X)


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15" | Enzyme
| align="center" | NA
| align="center" | NEB
| align="center" | NEB
| align="center" | Thermo
| align="center" | Thermo
| align="center" | Epi (1:10)
| align="center" | Epi (1:10)
| align="center" | NEB
| align="center" | NEB
| align="center" | NEB
| align="center" | NEB
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Circular Product (1X)
| height="15"  valign="bottom" | Circular Product (1X)
| align="center" align="center" valign="bottom" | 4
| align="center" align="center" valign="bottom" | 4
| align="center" align="center" valign="bottom" | 24.8
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 12.8
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 12.8
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 12.8
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 12.8
| align="center" align="center" valign="bottom" | 0
|- style="font-size:12pt"
| height="15"  valign="bottom" | Linear Product (1X)
| align="center" align="center" valign="bottom" | 4
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 24.8
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 12.8
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 12.8
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 12.8
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 12.8


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | RCA Primer (10 uM)
| height="15"  valign="bottom" | Bglii Cutter Primer (100 uM)
| align="center" align="center" valign="bottom" | 2.5
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 15.5
| align="center" align="center" valign="bottom" | 15.5
| align="center" align="center" valign="bottom" | 8
| align="center" align="center" valign="bottom" | 8
| align="center" align="center" valign="bottom" | 8
| align="center" align="center" valign="bottom" | 8
| align="center" align="center" valign="bottom" | 8
| align="center" align="center" valign="bottom" | 8
| align="center" align="center" valign="bottom" | 8
| align="center" align="center" valign="bottom" | 8


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | dNTP (1 mM)
| height="15"  valign="bottom" | 10X Buffer 3.1
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 12.4
| align="center" align="center" valign="bottom" | 12.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 10X Buffer
| height="15"  valign="bottom" | Bglii
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 12.4
| align="center" align="center" valign="bottom" | 12.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
| align="center" align="center" valign="bottom" | 6.4
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Phi29
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | BSA (10 mg/mL)
| align="center" align="center" valign="bottom" | 0.4
| align="center" align="center" valign="bottom" | 2.48
| align="center" align="center" valign="bottom" | 2.48
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 1.28
| align="center" align="center" valign="bottom" | 1.28
| align="center" align="center" valign="bottom" | 1.28
| align="center" align="center" valign="bottom" | 1.28


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 8.1
| align="center" align="center" valign="bottom" | 11
| align="center" align="center" valign="bottom" | 50.22
| align="center" align="center" valign="bottom" | 50.22
| align="center" align="center" valign="bottom" | 25.92
| align="center" align="center" valign="bottom" | 25.92
| align="center" align="center" valign="bottom" | 25.92
| align="center" align="center" valign="bottom" | 25.92
| align="center" align="center" valign="bottom" | 25.92
| align="center" align="center" valign="bottom" | 25.92
| align="center" align="center" valign="bottom" | 25.92
| align="center" align="center" valign="bottom" | 25.92


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Total
| height="15"  valign="bottom" | Total
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20
|style="font-weight:bold" align="center" align="center" valign="bottom" | 117.8
|style="font-weight:bold" align="center" align="center" valign="bottom" | 117.8
|style="font-weight:bold" align="center" align="center" valign="bottom" | 59.52
|style="font-weight:bold" align="center" align="center" valign="bottom" | 59.52
|style="font-weight:bold" align="center" align="center" valign="bottom" | 59.52
|style="font-weight:bold" align="center" align="center" valign="bottom" | 59.52
|style="font-weight:bold" align="center" align="center" valign="bottom" | 60.8
|style="font-weight:bold" align="center" align="center" valign="bottom" | 60.8
|style="font-weight:bold" align="center" align="center" valign="bottom" | 60.8
|style="font-weight:bold" align="center" align="center" valign="bottom" | 60.8


|}
|}


<li>Add 16.5 uL master mix to each tube</li>
<li>Use the following thermocycler program</li>
<li>Add 2.5 uL 10 uM RCA primer and to new reaction tubes according to following rules</li>
<ol type="a">
<ol type="a">
<li>"A" samples get LLRC</li>
<li>5 min 95C</li>
<li>"B" samples get LLRC-v03 (Mono-phosphorothiorate)</li>
<li>Ramp to 50C at 0.2C/s</li>
<li>"B" samples get LLRC-v04 (Tri-phosphorothiorate)</li></ol>
<li>10 min 50C</li>
<li>Heat reactions up to 95C for 5 min</li>
<li>4 hr 37C; '''When this cycle starts add 1 uL Bglii'''</li>
<li>Cool to 55C and incubate for 15 minutes</li>
<li>Heat kill with 65C for 20 min</li></ol>
<li>Cool to 30C and add enzyme</li>
</ol>
<li>Incubate at 30C for length of time according to table below</li>
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#CCFF66;font-size:12pt;font-weight:bold" align="center"
| width="85" height="32" | Sample
| width="105" | Enzyme
| width="110" | Incubation Time (hrs)


|- style="font-size:12pt"
Continued [[Daniel:Notebook/ComboLock/2017-3-26|tomorrow]]
| height="15"  valign="bottom" | X (ABC)
| align="center" valign="bottom" | NEB
| align="center" align="center" valign="bottom" | 3


|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Y (ABC)
| align="center" valign="bottom" | Thermo
| align="center" align="center" valign="bottom" | 3
|- style="font-size:12pt"
| height="15"  valign="bottom" | Z (ABC)
| align="center" valign="bottom" | Epi (1:10)
| align="center" align="center" valign="bottom" | 3
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | P (ABC)
| align="center" valign="bottom" | NEB
| align="center" align="center" valign="bottom" | 6
|- style="font-size:12pt"
| height="15"  valign="bottom" | Q (ABC)
| align="center" valign="bottom" | NEB
| align="center" align="center" valign="bottom" | 12
|}


<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol>


[[Category:ComboLock]] [[Category:20170324]]
[[Category:ComboLock]] [[Category:20170322]]

Latest revision as of 23:35, 30 March 2017

RCA Test[edit]

Back to Calendar

This experiment is to test the effectiveness of RCA so that I can use the most efficient method of amplification for the cell-based experiments. This builds on the T4 experiments and the circular product production. I will be using that circular product to analyze the effectiveness of RCA.

Linear Product Production (Continued from yesterday)[edit]

  1. TBE gel
    1. Mix 64 uL TBE and 16 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. File:GelLanes-20170325-LinearProduct.png
    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:2017-03-25-LinearProductProduction.png

Still too much circular product present. I'll try one more time to make it linear.

Linear Product Production[edit]

This time we'll start with the circular product and chop it.

  1. Bglii digest
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol
      Circular Product (1X) 4
      Bglii Cutter Primer (100 uM) 2
      10X Buffer 3.1 2
      Bglii 1
      nfH2O 11
      Total 20
    3. Use the following thermocycler program
      1. 5 min 95C
      2. Ramp to 50C at 0.2C/s
      3. 10 min 50C
      4. 4 hr 37C; When this cycle starts add 1 uL Bglii
      5. Heat kill with 65C for 20 min

    Continued tomorrow