Daniel:Notebook/ComboLock/2017-3-17: Difference between revisions

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>Djacobse
>Djacobse
Line 11: Line 11:
{| 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:#B1A0C7;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center"
| width="200" height="39" | Reagent
| width="200" height="28" | Reagent
| width="85" | Single Rxn
| width="85" | Single Rxn
| width="160" | MM1 (43.2X)
| width="150" | MM1 (23.2X)
| width="160" | MM+ (C1, 4.2X)
| width="150" | MM1-2 (20.2X)
| width="150" | MM+ (C1, 4.2X)


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Primer Pair (fwd/rev)
| height="15"  valign="bottom" | Primer Pair (fwd/rev)
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | primer12*/AmpR-Ind21
| align="center" valign="bottom" | AmpF/AmpR-Ind21
| align="center" valign="bottom" | AmpF/AmpR-Ind21
| align="center" valign="bottom" | primer24/primer4*
| align="center" valign="bottom" | primer24/primer4*


Line 25: Line 27:
| height="15"  valign="bottom" | Forward Primer (10 uM)
| height="15"  valign="bottom" | Forward Primer (10 uM)
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 15.2
| align="center" align="center" valign="bottom" | 23.2
| align="center" align="center" valign="bottom" | 20.2
| align="center" align="center" valign="bottom" | 4.2
| align="center" align="center" valign="bottom" | 4.2


Line 31: Line 34:
| height="15"  valign="bottom" | Reverse Primer (10 uM)
| height="15"  valign="bottom" | Reverse Primer (10 uM)
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 15.2
| align="center" align="center" valign="bottom" | 23.2
| align="center" align="center" valign="bottom" | 20.2
| align="center" align="center" valign="bottom" | 4.2
| align="center" align="center" valign="bottom" | 4.2


Line 37: Line 41:
| height="15"  valign="bottom" | nfH2O
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 21
| align="center" align="center" valign="bottom" | 21
| align="center" align="center" valign="bottom" | 319.2
| align="center" align="center" valign="bottom" | 487.2
| align="center" align="center" valign="bottom" | 424.2
| align="center" align="center" valign="bottom" | 88.2
| align="center" align="center" valign="bottom" | 88.2


Line 43: Line 48:
| height="15"  valign="bottom" | 2X Kapa SYBR Fast
| height="15"  valign="bottom" | 2X Kapa SYBR Fast
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 380
| align="center" align="center" valign="bottom" | 580
| align="center" align="center" valign="bottom" | 505
| align="center" align="center" valign="bottom" | 105
| align="center" align="center" valign="bottom" | 105


Line 49: Line 55:
| height="15"  valign="bottom" | Total
| height="15"  valign="bottom" | Total
|style="font-weight:bold" align="center" align="center" valign="bottom" | 48
|style="font-weight:bold" align="center" align="center" valign="bottom" | 48
|style="font-weight:bold" align="center" align="center" valign="bottom" | 729.6
|style="font-weight:bold" align="center" align="center" valign="bottom" | 1113.6
|style="font-weight:bold" align="center" align="center" valign="bottom" | 969.6
|style="font-weight:bold" align="center" align="center" valign="bottom" | 201.6
|style="font-weight:bold" align="center" align="center" valign="bottom" | 201.6



Revision as of 16:01, 17 March 2017

Circularization Comparison (T4 Poly/Ligase; Started Yesterday)

Back to Calendar

Protocol-Part 6

  • qPCR
    1. Make qPCR master mixes according to following recipe
    2. Reagent Single Rxn MM1 (23.2X) MM1-2 (20.2X) MM+ (C1, 4.2X)
      Primer Pair (fwd/rev) NA AmpF/AmpR-Ind21 AmpF/AmpR-Ind21 primer24/primer4*
      Forward Primer (10 uM) 1 23.2 20.2 4.2
      Reverse Primer (10 uM) 1 23.2 20.2 4.2
      nfH2O 21 487.2 424.2 88.2
      2X Kapa SYBR Fast 25 580 505 105
      Total 48 1113.6 969.6 201.6
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. File:PlateLayout-20170317-T4Circ-PostRCA.png
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  • TBE Gel
    1. Mix 200 uL TBE and 50 uL 6X dye
    2. Aliquot 10 uL per sample on to parafilm
    3. Add 2 uL sample or 1.5 uL ladder to aliquots
    4. Mix and add 10 uL per lane to gel
    5. Run gel at 230V for 24 minutes
    6. Add 2 uL SYBR gold and incubate on shaker for 3 minutes
    7. Rinse and image in gel doc