Daniel:Notebook/ComboLock/2016-8-17: Difference between revisions
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===Nanodrop Results=== | |||
=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" | | |||
| align="center" valign="bottom" | X | |||
| align="center" align="center" valign="bottom" | | |||
| 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" | | |||
| align="center" valign="bottom" | X | |||
| align="center" align="center" valign="bottom" | | |||
| 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" | | |||
| 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" | | |||
| 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" | | |||
| align="center" align="center" valign="bottom" | | |||
| 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" | | |||
| align="center" align="center" valign="bottom" | | |||
| 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 22:47, 17 August 2016
Positive Control Amplicon Production (Started yesterday)
Protocol
- Size select gel
- Mix 30 uL TBE, 6 uL loading dye, and 4 uL sample for C1-C2 and C3-C2
- Mix 15 uL TBE, 3 uL loading dye, and 2 uL ladder
- Add 20 uL to each lane (2 lanes for C1-C2 and C3-C2)
- Run the gel at 250V for 25 min
- Stain with 3 uL SYBR gold for 3 min
- Image, and extract bands between 100bp and 125 bp; collect the gel bands in the same tube
- 2016-08-17-PosControlAmplicon-SizeSelect.png
Before Image
- 2016-08-17-PosControlAmplicon-SizeSelect-After.png
After Image
- Shred the gel by centrifuging at 14000rpm for 1 min 30 sec
- Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking
- Centrifuge at 12000rpm for 1.5 min
- Extract supernatant and run through a nanosep column; centrifuge for 1.5 min at 12000rpm
- Ethanol Precipitation
- Move flow through to a 2 mL tube
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue
- Incubate at -80C for 4 hours
- Pellet by centrifuging for 25 min at 12000rpm at 4C
- Remove supernatant and add 750 uL chilled 70% EtOH
- Centrifuge for 12 min at 12000 rpm and 4 C
- Remove supernatant and air dry
- Resuspend in 30 uL nfH2O and measure in nanodrop
- Latch and Padlock Hybridization
- Mix together samples according to table; use 100 uL LPH buffer with 100 nM Padlock0001-68
- Incubate for 1 hr at 37C
- Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
- Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
- For sample 3, use 10 uL 100 uM Latch0001 (10 uM final concentration)
- Incubate for 30 min at 37C
- Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
- Circularization
- Prepare 20 uL KLN mix
- Prepare 4.1x master mix of Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
- Add 2 uL KLN mix to 20 uL of reaction for sample 1
- Incubate at 55C overnight (XX hours)
- Continued tomorrow
Nanodrop Results
Positive Control Amplicon Test
Sample No | Sample Name | LatchX2 | LatchX3 | PCAmp1 | PCAmp3 | Expected Product |
1 | Amp1 Test | X | X | Amp1 | ||
2 | Amp3 Test | X | X | Amp3 | ||
3 | LatchX2 Specificity | X | X | X | Amp1 | |
4 | LatchX3 Specificity | X | X | X | Amp3 | |
5 | No Template | X | X | None | ||
6 | No Latch | X | X | None | ||
7 | No Padlock | X | X | X | X | None |