Daniel:Notebook/ComboLock/2017-4-3: Difference between revisions
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(14 intermediate revisions by the same user not shown) | |||
Line 27: | Line 27: | ||
<li>Goto (b) x20</li> | <li>Goto (b) x20</li> | ||
<li>72C for 2 min</li></ol> | <li>72C for 2 min</li></ol> | ||
</ol> | </ol></ol> | ||
< | |||
==Protocol-[[Daniel:Notebook/ComboLock/2017-3-29|Post-Bglii samples]]== | |||
<ol start="2"> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | <ol type="A"> | ||
<li>Mix 80 uL TBE and 20 uL 6X dye</li> | <li>Mix 80 uL TBE and 20 uL 6X dye</li> | ||
Line 38: | Line 42: | ||
<li>Rinse and image in gel doc</li> | <li>Rinse and image in gel doc</li> | ||
</ol> | </ol> | ||
<li>Qiaquick Column | <li>Qiaquick Column</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Add 200 uL (5X) PB buffer to 40 uL sample</li> | <li>Add 200 uL (5X) PB buffer to 40 uL sample</li> | ||
Line 51: | Line 55: | ||
</ol> | </ol> | ||
<li>TBE Gel</li> | <li>TBE Gel</li> | ||
<li> | <ol type="A"> | ||
<li>Mix 40 uL 10X TBE and 20 uL 6X dye</li> | |||
<li>Aliquot 10 uL on to parafilm per sample, and 10 uL TBE ladder</li> | |||
<li>Add 6 uL reaction or 1.5 uL ladder to appropriate aliquot</li> | |||
<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> | |||
===Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:20170403-qPCR-RCATest-CPLP-ssSynthesis.png|qPCR curves | |||
File:2017-04-03-RCATest-CPLP-ssSynthesis-PostBglii.png|Pre-purification gel | |||
File:2017-04-03-RCATest-CPLP-ssSynthesis-PostBglii-Purified.png|Post purification gel | |||
</gallery> | |||
Purification really didn't do much but it the gels look good. The product should be around 200bp, btw, because I only used AmpF, which adds on about 20bp | |||
==Protocol-[[Daniel:Notebook/ComboLock/2017-3-26|Pre-Bglii samples]]== | |||
<ol start="2"> | |||
<li>Bgl Digestion</li> | |||
<ol type="A"> | <ol type="A"> | ||
<li>Make the following reactions; '''Do not add Bglii yet'''</li> | <li>Make the following reactions; '''Do not add Bglii yet'''</li> | ||
Line 57: | Line 84: | ||
{| 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="font-size:12pt;font-weight:bold" align="center" | |- style="font-size:12pt;font-weight:bold" align="center" | ||
|style="background-color:#8DB4E2" width=" | |style="background-color:#8DB4E2" width="210" height="42" | Reagent | ||
|style="background-color:#8DB4E2" width="85" | Single Rxn Vol | |style="background-color:#8DB4E2" width="85" | Single Rxn Vol | ||
|style="background-color:#95B3D7" width=" | |style="background-color:#95B3D7" width="85" | Master Mix (6.2X) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | RCA Rxn | | height="15" valign="bottom" | RCA Rxn | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 8 | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | | | 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" | | | align="center" align="center" valign="bottom" | 12.4 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Bglii | | height="15" valign="bottom" | Bglii | ||
| 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 | ||
|- style="font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 9 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 55.8 | ||
|- style=" | |- style="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" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 68.2 | ||
|} | |} | ||
Line 98: | Line 120: | ||
<li>Ramp to 50C at 0.2C/s</li> | <li>Ramp to 50C at 0.2C/s</li> | ||
<li>10 min 50C</li> | <li>10 min 50C</li> | ||
<li> | <li>1 hr 37C; '''When this cycle starts add 1 uL Bglii'''</li> | ||
<li>Heat kill with 65C for 20 min</li></ol> | <li>Heat kill with 65C for 20 min</li></ol> | ||
</ol> | </ol> | ||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 60 uL 10X TBE and 20 uL 6X dye</li> | |||
<li>Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot</li> | |||
<li>Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot</li> | |||
<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:2017-04-04_-RCATest-CPLP-ssSynthesis-sssThenBgl.png|500px]] | |||
It appears that the Bgl digestion was incomplete. That does mean there is a lot more product available than can be cut in an hour with one uL of Bglii, which according to [https://www.neb.com/products/r0144-bglii#pd-protocols NEB] is 10 ug (1 ug/unit/hr) | |||
[[Category:ComboLock]] [[Category:20170324]] | [[Category:ComboLock]] [[Category:20170324]] |
Latest revision as of 16:17, 7 April 2017
RCA Test (Started March 24)[edit]
I'll try qPCR on the RCA product again, this time on the NEB 12 hr samples (since I am running low on X samples), but for few cycles and with only the forward primer (which should be RC to the RCA product. This will be fed into Bglii digestion
- qPCR-Second strand synthesis
- Make the following 13.2X master mix
- 13.2 uL AmpF-6.4Sol (10 uM)
- 264 uL Kapa SYBR 2X Master Mix
- 224.4 uL nfH2O
- Aliquot 38 uL master mix into each lane
- Add 2 uL sample according to plate layout File:PlateLayout-20170403-RCATest-CPLP-ssSynthesis.png
- Run the following protocol
- 95C for 2 min
- 95C for 5 sec
- 55C for 30 sec
- 72C for 45 sec
- Goto (b) x20
- 72C for 2 min
Protocol-Post-Bglii samples[edit]
- TBE Gel
- Mix 80 uL TBE and 20 uL 6X dye
- Aliquot 10 uL on to parafilm per sample/ladder
- Add 2.5 uL reaction or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
- Qiaquick Column
- Add 200 uL (5X) PB buffer to 40 uL sample
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
- Dry spin column for 1 minute at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Add 35 uL nfH2O to column
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- TBE Gel
- Mix 40 uL 10X TBE and 20 uL 6X dye
- Aliquot 10 uL on to parafilm per sample, and 10 uL TBE ladder
- Add 6 uL reaction or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
Results[edit]
- 20170403-qPCR-RCATest-CPLP-ssSynthesis.png
qPCR curves
- 2017-04-03-RCATest-CPLP-ssSynthesis-PostBglii.png
Pre-purification gel
- 2017-04-03-RCATest-CPLP-ssSynthesis-PostBglii-Purified.png
Post purification gel
Purification really didn't do much but it the gels look good. The product should be around 200bp, btw, because I only used AmpF, which adds on about 20bp
Protocol-Pre-Bglii samples[edit]
- Bgl Digestion
- Make the following reactions; Do not add Bglii yet
- Use the following thermocycler program
- 5 min 95C
- Ramp to 50C at 0.2C/s
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- TBE Gel
- Mix 60 uL 10X TBE and 20 uL 6X dye
- Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot
- Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
Reagent | Single Rxn Vol | Master Mix (6.2X) |
RCA Rxn | 8 | 0 |
10X Buffer 3.1 | 2 | 12.4 |
Bglii | 1 | 0 |
nfH2O | 9 | 55.8 |
Total | 20 | 68.2 |
File:2017-04-04 -RCATest-CPLP-ssSynthesis-sssThenBgl.png
It appears that the Bgl digestion was incomplete. That does mean there is a lot more product available than can be cut in an hour with one uL of Bglii, which according to NEB is 10 ug (1 ug/unit/hr)