Daniel:Notebook/ComboLock/2016-8-17: Difference between revisions

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=Positive Control Amplicon Test=
<ol>
<li>Latch and Padlock Hybridization</li>
<ol type="A">
<li>Mix together samples according to table; use 100 uL LPH buffer with 100 nM Padlock0001-68 </li>
<li>Incubate for 1 hr at 37C</li>
<li>Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min</li>
<li>Incubate cells for 20 min at 40C in 150 uL stringent wash buffer</li>
<li>Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001</li>
<ol type="a"><li>For sample 3, use 10 uL 100 uM Latch0001 (10 uM final concentration)</li></ol>
<li>Incubate for 30 min at 37C</li>
<li>Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min</li>
<li>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li>
<li>Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube</li></ol>
<li>Circularization</li>
<ol type="A">
<li>Prepare 20 uL KLN mix</li>
<li>Prepare 4.1x master mix of Phusion mix</li>
<li>Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5</li>
<li>Add 2 uL KLN mix to 20 uL of reaction for sample 1</li>
<li>Incubate at 55C overnight (XX hours) </li>
<li>Continued [[Daniel:Notebook/ComboLock/2016-8-18|tomorrow]]</li>
</ol>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center"
| width="42" height="31" | Sample No
| width="121" | Sample Name
| width="54" | LatchX2
| width="53" | LatchX3
| width="54" | PCAmp1
| width="51" | PCAmp3
| width="100" | Expected Product
|- style="font-size:12pt"
| align="center" height="15" valign="bottom" | 1
|  valign="bottom" | Amp1 Test
| align="center" valign="bottom" | X
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" valign="bottom" | X
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" valign="bottom" | Amp1
|- style="background-color:#D9D9D9;font-size:12pt"
| align="center" height="15" valign="bottom" | 2
|  valign="bottom" | Amp3 Test
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" valign="bottom" | X
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" valign="bottom" | X
| align="center" valign="bottom" | Amp3
|- style="font-size:12pt"
| align="center" height="15" valign="bottom" | 3
|  valign="bottom" | LatchX2 Specificity
| align="center" valign="bottom" | X
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | Amp1
|- style="background-color:#D9D9D9;font-size:12pt"
| align="center" height="15" valign="bottom" | 4
|  valign="bottom" | LatchX3 Specificity
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | Amp3
|- style="font-size:12pt"
| align="center" height="15" valign="bottom" | 5
|  valign="bottom" | No Template
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" valign="bottom" | None
|- style="background-color:#D9D9D9;font-size:12pt"
| align="center" height="15" valign="bottom" | 6
|  valign="bottom" | No Latch
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | None
|- style="font-size:12pt"
| align="center" height="15" valign="bottom" | 7
|  valign="bottom" | No Padlock
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | None
|}
[[Category:ComboLock]] [[Category:20160805]]
[[Category:ComboLock]] [[Category:20160805]]

Revision as of 15:14, 18 August 2016

Positive Control Amplicon Production (Started yesterday)

Back to Calendar

Protocol

  1. Size select gel
    1. Mix 30 uL TBE, 6 uL loading dye, and 4 uL sample for C1-C2 and C3-C2
    2. Mix 15 uL TBE, 3 uL loading dye, and 2 uL ladder
    3. Add 20 uL to each lane (2 lanes for C1-C2 and C3-C2)
    4. Run the gel at 250V for 25 min
    5. Stain with 3 uL SYBR gold for 3 min
    6. Image, and extract bands between 100bp and 125 bp; collect the gel bands in the same tube
    7. Shred the gel by centrifuging at 14000rpm for 1 min 30 sec
    8. Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking
    9. Centrifuge at 12000rpm for 1.5 min
    10. Extract supernatant and run through a nanosep column; centrifuge for 1.5 min at 12000rpm
  2. Ethanol Precipitation
    1. Move flow through to a 2 mL tube
    2. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue
    3. Incubate at -80C for 4 hours
    4. Pellet by centrifuging for 25 min at 12000rpm at 4C
    5. Remove supernatant and add 750 uL chilled 70% EtOH
    6. Centrifuge for 12 min at 12000 rpm and 4 C
    7. Remove supernatant and air dry
    8. Resuspend in 30 uL nfH2O and measure in nanodrop

    Nanodrop Results