Revision as of 12:38, 4 November 2016

UCONN iGEM

Notebook

October 13, 2016

Transformed 5uL of BBa_J04450 at a concentration of 20pg/uL into Stellar competent cells using the following protocol:

Remove competent cells from -80°C freezer and thaw on wet ice (10-15 minutes).
Add DNA (pre-chilled) to 100μL of cells on ice. Stir briefly with a pipet tip – DO NOT pipet up and down to mix as this can heat up cells and introduce bubbles.
Incubate on ice for 30 minutes.
Heat-shock cells by placing tube in a 37°C heat block for 1 min.
Return cells to ice for 2 minutes.
Add 950μL of room temperature SOC to the cells in the culture tube.
Place tubes in a shaking incubator at 250 rpm for 1 hour at 37°C.
Centrifuge at 1,000g for 1 min, remove and discard 950μL of supernatant, resuspending cells gently in remaining SOC (~150μL).
Plate entire sample of transformed cells on LB agar plates with the chloramphenicol.
Incubate plates upside down overnight at 37°C.

October 14, 2016

Colonies from previous transformation were selected on the criteria of size and isolation for overnight culture. The selected colonies were removed with a pipette tip. The pipette tip was then placed into a 14mL culture tubes containing 10mL LB and 10uL 1000X Chloramphenicol. The plates were incubated overnight at 37°C.

October 15, 2016

Verification of BBa_J04450 via PCR

Master Mix

20µL Phusion Buffer

2µL Primers

2µL dNTP

Primer Dilution

1 µL SB-Prep Forward Primer

1 µL SB-Prep Reverse Primer

2 µL PCR Grade H2O

Setup 2 50µL Reactions:

PCR A

1µL DNA (from mini prep of overnight culture A)

12µL Master Mix

0.5 µL Phusion

36.5µL PCR Grade H2O

PCR B

2µL DNA (from mini prep of overnight culture B)

12µL Master Mix

0.5 µL Phusion

35.5 PCR grade H2O

Amplify with the following program:

98°C for 30s

35 Cycles of:

98°C for 10s

47-65°C for 30s

72°C for 30-45s/1kb

72°C for 10 minutes

12°C hold

PCR Products were analyzed using the gel electrophoresis protocol that follows on October 16, 2016.

October 16, 2016

Digest of Vector and Insert

Set up 2 20uL reactions:

Vector

5µL 10X Cutsmart Buffer

1µL NotI

14µL vector DNA

17µL ddH2O

Insert

5µL 10X Cutsmart Buffer

1µL NotI

29.2µL trkH DNA

14.9µL ddH2O

Incubate in thermocycler using the following program:

Incubate in thermocycler as follows:

37°C for 2 hours
12°C hold

Add 1μL Calf Intestinal Phosphatase to the vector reaction only and incubate at 37°C for 1 hour.

Digest products were analyzed using the following protocol:

Gel Electrophoresis Protocol

Add to 250mL Erlenmeyer flask

0.5g Agarose
50mL TAE

Heat in microwave until completely dissolved. Do not allow liquid to boil over, remove and swirl periodically.
Add 5μL Gel Green
Pour into gel case (make sure to eliminate bubbles with pipette tip) and insert comb. Allow gel to set. Can be left at 4°C to accelerate setting, or stored (wrapped in plastic) for up to 1 week.
Place gel into gel box and cover with TAE.
Add 10uL 6X loading dye to each PCR reaction and load 12uL of each.
Load 10μL TriDye 2-Log Ladder to the first lane.
Run until dye front reaches the end of the gel at 100V (about 75 min).
Image on ChemiDoc using either Ethidium Bromide or Gel Green program.

The gel was loaded as follows

10uL TriDye 2-Log Ladder
Lanes 2-4 17uL Digested Insert
Lanes 6-8 17uL Digested Vector

The bands of interest were excised based on size and purified using the following protocol.

Gel Purification of Digest

Weigh gel slice.
Place a SV Minicolumn into a collection tube.
Add Membrane Binding Solution at a ratio of 10μl of solution per 10mg of agarose gel slice.
Vortex (or invert) the mixture and incubate at 55°C for 10 minutes or until the gel slice is completely dissolved.
Transfer up to 350μL dissolved gel sample to column and incubate 3 min at room temperature.
Centrifuge 16,000g for 1 min and discard flowthrough.
Add 700μL Membrane Wash solution, centrifuge 16,000g for 1 min and discard flowthrough.
Perform second was with 500μL Membrane Wash solution, centrifuge 16,000g for 5 min and discard flowthrough.
Dry membrane by centrifuging 16,000g for 1 min.
Transfer column to new, sterile 1.5mL tube and add 23.5μL Nuclease-free water. Incubate at room temperature for 4 min.
Centrifuge at 16,000g for 1 min.
Determine DNA concentration by NanoDrop.

DpnI Digest of BBa_J04450 for Fast Cloning

1µL DpnI

2µL Cutsmart Buffer

15µL PCR products (*PCR products not cleaned)

2µL ddH2O

Incubate in the thermocycler with the following program:

37C for 2hrs

80C for 20 mins

4C Forever

Fast Cloning

DpnI Digest of BBa_J04450 and trkH were transformed into stellar cells using the aforementioned transformation protocol in the following volumetric ratios:

1uL vector : 1uL insert

1uL vector : 2uL insert

1uL vector : 3uL insert

October 17, 2016

Ligation of Vector and Insert

Set up 1 50µL reaction:

5µL 10X T4 ligase buffer

20µL Insert

6.19µL Vector

1µL DNA Ligase

17.81µL ddH2O

Incubate in the thermocycler with the following protocol:

45oC 5 mins

16oC overnight

24oC 30 mins

Overnight Culture

Colonies from the fast cloning transformation were selected from each of the different dilutions. Overnight cultures were inoculated using the aforementioned culture protocol.

October 18, 2016

Verification of Insert PCR

Master Mix

30µL Buffer

3µL Primers

3µL dNTP

Primer Dilution

2 µL VF2 Primer

2 µL VR Primer

4 µL PCR Grade H2O

All DNA was diluted to 50ng/uL

Setup 3 50µL Reactions:

PCR 1:1 Dilution

1µL DNA

12µL Master Mix

0.5 µL Phusion

36.5µL PCR Grade H2O

PCR 1:2 Dilution

2µL DNA

12µL Master Mix

0.5 µL Phusion

36.5 PCR grade H2O

PCR 1:3 Dilution

1µL DNA

12µL Master Mix

0.5 µL Phusion

36.5µL PCR Grade H2O

Amplify with the following program:

98°C for 30s

35 Cycles of:

98°C for 10s

47-65°C for 30s

72°C for 30-45s/1kb

72°C for 10 minutes

12°C hold

PCR Products were analyzed using the aforementioned gel electrophoresis protocol. The gel was loaded with 12uL of each sample and 10uL of ladder.

Ladder
1:1 Dilution
1:2 Dilution
1:3 Dilution

Transformation of Ligation Products

The ligation products were transformed into Stellar cells at a volume of 20uL using the aforementioned transformation protocol.

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class="c2"><span class="c0">October 13, &nbsp;2016</span></p><p class="c1"><span class="c0"></span></p><p class="c2"><span class="c3">Transformed 5uL of BBa_J04450 at a concentration of 20pg/uL into Stellar competent cells using the following protocol: </span></p><p class="c1"><span class="c3"></span></p><ol class="c14 lst-kix_r2uxxnh679pj-0 start" start="1"><li class="c2 c4"><span class="c3">Remove competent cells from -80&deg;C freezer and thaw on wet ice (10-15 minutes). </span></li><li class="c2 c4"><span class="c3">Add DNA (pre-chilled) to 100&mu;L of cells on ice. &nbsp;Stir briefly with a pipet tip &ndash; </span><span class="c0">DO NOT</span><span class="c3">&nbsp;pipet up and down to mix as this can heat up cells and introduce bubbles.</span></li><li class="c2 c4"><span class="c3">Incubate on ice for 30 minutes.</span></li><li class="c2 c4"><span class="c3">Heat-shock cells by placing tube in a 37&deg;C heat block for 1 min.</span></li><li class="c2 c4"><span class="c3">Return cells to ice for 2 minutes.</span></li><li class="c2 c4"><span class="c3">Add 950&mu;L of room temperature SOC to the cells in the culture tube.</span></li><li class="c2 c4"><span class="c3">Place tubes in a shaking incubator at 250 rpm for 1 hour at 37&deg;C.</span></li><li class="c2 c4"><span class="c3">Centrifuge at 1,000g for 1 min, remove and discard 950&mu;L of supernatant, resuspending cells gently in remaining SOC (~150&mu;L).</span></li><li class="c2 c4"><span class="c3">Plate entire sample of transformed cells on LB agar plates with the chloramphenicol.</span></li><li class="c2 c4"><span class="c3">Incubate plates upside down overnight at 37&deg;C.</span></li></ol><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c0">October 14, 2016</span></p><p class="c1"><span class="c0"></span></p><p class="c2"><span class="c3">Colonies from previous transformation were selected on the criteria of size and isolation for overnight culture. &nbsp;The selected colonies were removed with a pipette tip. &nbsp;The pipette tip was then placed into a 14mL culture tubes containing 10mL LB and 10uL 1000X Chloramphenicol. &nbsp;The plates were incubated overnight at 37&deg;C. &nbsp;</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c0">October 15, 2016</span></p><p class="c1"><span class="c0"></span></p><p class="c2"><span class="c3">Verification of BBa_J04450 via PCR</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">Master Mix</span></p><p class="c2"><span class="c3">20&micro;L Phusion Buffer</span></p><p class="c2"><span class="c3">2&micro;L Primers</span></p><p class="c2"><span class="c3">2&micro;L dNTP</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">Primer Dilution</span></p><p class="c2"><span class="c3">1 &micro;L SB-Prep Forward Primer</span></p><p class="c2"><span class="c3">1 &micro;L SB-Prep Reverse Primer</span></p><p class="c2"><span class="c3">2 &micro;L PCR Grade H</span><span class="c12">2</span><span class="c3">O</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">Setup 2 50&micro;L Reactions: </span></p><p class="c2"><span class="c3 c7">PCR A </span></p><p class="c2"><span class="c3">1&micro;L DNA (from mini prep of overnight culture A)</span></p><p class="c2"><span class="c3">12&micro;L Master Mix</span></p><p class="c2"><span class="c3">0.5 &micro;L Phusion</span></p><p class="c2"><span class="c3">36.5&micro;L PCR Grade H</span><span class="c12">2</span><span class="c3">O</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3 c7">PCR B</span></p><p class="c2"><span class="c3">2&micro;L DNA (from mini prep of overnight culture B)</span></p><p class="c2"><span class="c3">12&micro;L Master Mix</span></p><p class="c2"><span class="c3">0.5 &micro;L Phusion </span></p><p class="c2"><span class="c3">35.5 PCR grade H</span><span class="c12">2</span><span class="c3">O</span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">Amplify with the following program:</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">98&deg;C for 30s</span></p><p class="c2"><span class="c3">35 Cycles of:</span></p><p class="c2"><span class="c3">98&deg;C for 10s</span></p><p class="c2"><span class="c3">47-65&deg;C for 30s</span></p><p class="c2"><span class="c3">72&deg;C for 30-45s/1kb</span></p><p class="c2"><span class="c3">72&deg;C for 10 minutes</span></p><p class="c2"><span class="c3">12&deg;C hold</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">PCR Products were analyzed using the gel electrophoresis protocol that follows on October 16, 2016. &nbsp;</span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c0">October 16, 2016</span></p><p class="c1"><span class="c0"></span></p><p class="c2"><span class="c0">Digest of Vector and Insert</span></p><p class="c2"><span class="c3">Set up 2 20uL reactions:</span></p><p class="c2"><span class="c3">Vector</span></p><p class="c2"><span class="c3">5&micro;L 10X Cutsmart Buffer</span></p><p class="c2"><span class="c3">1&micro;L NotI</span></p><p class="c2"><span class="c3">14&micro;L vector DNA</span></p><p class="c2"><span class="c3">17&micro;L ddH2O</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">Insert</span></p><p class="c2"><span class="c3">5&micro;L 10X Cutsmart Buffer</span></p><p class="c2"><span class="c3">1&micro;L NotI</span></p><p class="c2"><span class="c3">29.2&micro;L trkH DNA</span></p><p class="c2"><span class="c3">14.9&micro;L ddH2O</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">Incubate in thermocycler using the following program: </span></p><ol class="c14 lst-kix_72bw7pq3eyoo-0 start" start="1"><li class="c2 c4"><span class="c3">Incubate in thermocycler as follows: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c14 lst-kix_72bw7pq3eyoo-1 start" start="1"><li class="c2 c9"><span class="c3">37&deg;C for 2 hours &nbsp;</span></li><li class="c2 c9"><span class="c3">12&deg;C hold</span></li></ol><ol class="c14 lst-kix_72bw7pq3eyoo-0" start="2"><li class="c2 c4"><span class="c3">Add 1&mu;L Calf Intestinal Phosphatase to the vector reaction only</span><span class="c0">&nbsp;</span><span class="c3">and incubate at 37&deg;C for 1 hour.</span></li></ol><p class="c1 c15"><span class="c3"></span></p><p class="c2"><span class="c3">Digest products were analyzed using the following protocol:</span></p><p class="c2"><span class="c3">Gel Electrophoresis Protocol </span></p><ol class="c14 lst-kix_x809dwpv41f6-0 start" start="1"><li class="c2 c4"><span class="c3">Add to 250mL Erlenmeyer flask</span></li></ol><ol class="c14 lst-kix_x809dwpv41f6-1 start" start="1"><li class="c2 c9"><span class="c3">0.5g Agarose</span></li><li class="c2 c9"><span class="c3">50mL TAE</span></li></ol><ol class="c14 lst-kix_x809dwpv41f6-0" start="2"><li class="c2 c4"><span class="c3">Heat in microwave until completely dissolved. &nbsp;Do not allow liquid to boil over, remove and swirl periodically.</span></li><li class="c2 c4"><span class="c3 c10">Add 5</span><span class="c3">&mu;L Gel Green</span></li><li class="c2 c4"><span class="c3">Pour into gel case (make sure to eliminate bubbles with pipette tip) and insert comb. &nbsp;Allow gel to set. &nbsp;Can be left at 4&deg;C to accelerate setting, or stored (wrapped in plastic) for up to 1 week.</span></li><li class="c2 c4"><span class="c3">Place gel into gel box and cover with TAE.</span></li><li class="c2 c4"><span class="c3">Add 10uL 6X loading dye to each PCR reaction and load 12uL of each.</span></li><li class="c2 c4"><span class="c3">Load 10&mu;L TriDye 2-Log Ladder to the first lane.</span></li><li class="c2 c4"><span class="c3">Run until dye front reaches the end of the gel at 100V (about 75 min)</span><span class="c3 c8">.</span></li><li class="c2 c4"><span class="c3">Image on ChemiDoc using either Ethidium Bromide or Gel Green program.</span></li></ol><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">The gel was loaded as follows</span></p><ol class="c14 lst-kix_2ix42o5x5fps-0 start" start="1"><li class="c2 c4"><span class="c3">10uL TriDye 2-Log Ladder</span></li><li class="c2 c4"><span class="c3">Lanes 2-4 17uL Digested Insert</span></li><li class="c2 c4"><span class="c3">Lanes 6-8 17uL Digested Vector</span></li></ol><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">The bands of interest were excised based on size and purified using the following protocol. &nbsp;</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c0">Gel Purification of Digest&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></p><ol class="c14 lst-kix_4rw8lxud3ivq-0 start" start="1"><li class="c2 c4"><span class="c3">Weigh gel slice.</span></li><li class="c2 c4"><span class="c3">Place a SV Minicolumn into a collection tube.</span></li><li class="c2 c4"><span class="c3">Add Membrane Binding Solution at a ratio of 10&mu;l of solution per 10mg of agarose gel slice.</span></li><li class="c2 c4"><span class="c3">Vortex (or invert) the mixture and incubate at 55&deg;C for 10 minutes or until the gel slice is completely dissolved.</span></li><li class="c2 c4"><span class="c3">Transfer up to 350&mu;L dissolved gel sample to column and incubate 3 min at room temperature.</span></li><li class="c2 c4"><span class="c3">Centrifuge 16,000g for 1 min and discard flowthrough.</span></li><li class="c2 c4"><span class="c3">Add 700&mu;L Membrane Wash solution, centrifuge 16,000g for 1 min and discard flowthrough.</span></li><li class="c2 c4"><span class="c3">Perform second was with 500&mu;L Membrane Wash solution, centrifuge 16,000g for 5 min and discard flowthrough.</span></li><li class="c2 c4"><span class="c3">Dry membrane by centrifuging 16,000g for 1 min.</span></li><li class="c2 c4"><span class="c3">Transfer column to new, sterile 1.5mL tube and add 23.5&mu;L Nuclease-free water. &nbsp;Incubate at room temperature for 4 min.</span></li><li class="c2 c4"><span class="c3">Centrifuge at 16,000g for 1 min.</span></li><li class="c2 c4"><span class="c3">Determine DNA concentration by NanoDrop.</span></li></ol><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c0">DpnI Digest of BBa_J04450 for Fast Cloning</span></p><p class="c2"><span class="c3">1&micro;L DpnI</span></p><p class="c2"><span class="c3">2&micro;L Cutsmart Buffer</span></p><p class="c2"><span class="c3">15&micro;L PCR products (*PCR products not cleaned)</span></p><p class="c2"><span class="c3">2&micro;L ddH2O</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">Incubate in the thermocycler with the following program:</span></p><p class="c2"><span class="c3">37C for 2hrs</span></p><p class="c2"><span class="c3">80C for 20 mins</span></p><p class="c2"><span class="c3">4C Forever</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c0">Fast Cloning</span></p><p class="c2"><span class="c3">DpnI Digest of BBa_J04450 and trkH were transformed into stellar cells using the aforementioned transformation protocol in the following volumetric ratios:</span></p><p class="c2"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1uL vector : 1uL insert</span></p><p class="c2"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1uL vector : 2uL insert</span></p><p class="c2"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1uL vector : 3uL insert</span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c0">October 17, 2016</span></p><p class="c1"><span class="c0"></span></p><p class="c2"><span class="c0">Ligation of Vector and Insert </span></p><p class="c2"><span class="c3">Set up 1 50&micro;L reaction:</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">5&micro;L 10X T4 ligase buffer</span></p><p class="c2"><span class="c3">20&micro;L Insert</span></p><p class="c2"><span class="c3">6.19&micro;L Vector</span></p><p class="c2"><span class="c3">1&micro;L DNA Ligase</span></p><p class="c2"><span class="c3">17.81&micro;L ddH2O</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">Incubate in the thermocycler with the following protocol:</span></p><p class="c2"><span class="c3">45</span><span class="c11">o</span><span class="c3">C 5 mins</span></p><p class="c2"><span class="c3">16</span><span class="c11">o</span><span class="c3">C overnight</span></p><p class="c2"><span class="c3">24</span><span class="c11">o</span><span class="c3">C 30 mins</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c0">Overnight Culture</span></p><p class="c2"><span class="c3">Colonies from the fast cloning transformation were selected from each of the different dilutions. Overnight cultures were inoculated using the aforementioned culture protocol. &nbsp;</span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c0">October 18, 2016</span></p><p class="c1"><span class="c0"></span></p><p class="c2"><span class="c0">Verification of Insert PCR</span></p><p class="c2"><span class="c3">Master Mix</span></p><p class="c2"><span class="c3">30&micro;L Buffer</span></p><p class="c2"><span class="c3">3&micro;L Primers</span></p><p class="c2"><span class="c3">3&micro;L dNTP</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">Primer Dilution</span></p><p class="c2"><span class="c3">2 &micro;L VF2 Primer</span></p><p class="c2"><span class="c3">2 &micro;L VR Primer</span></p><p class="c2"><span class="c3">4 &micro;L PCR Grade H</span><span class="c12">2</span><span class="c3">O</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">All DNA was diluted to 50ng/uL</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">Setup 3 50&micro;L Reactions: </span></p><p class="c2"><span class="c3 c7">PCR 1:1 Dilution</span></p><p class="c2"><span class="c3">1&micro;L DNA</span></p><p class="c2"><span class="c3">12&micro;L Master Mix</span></p><p class="c2"><span class="c3">0.5 &micro;L Phusion</span></p><p class="c2"><span class="c3">36.5&micro;L PCR Grade H</span><span class="c12">2</span><span class="c3">O</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3 c7">PCR 1:2 Dilution</span></p><p class="c2"><span class="c3">2&micro;L DNA </span></p><p class="c2"><span class="c3">12&micro;L Master Mix</span></p><p class="c2"><span class="c3">0.5 &micro;L Phusion </span></p><p class="c2"><span class="c3">36.5 PCR grade H</span><span class="c12">2</span><span class="c3">O</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">PCR 1:3 Dilution</span></p><p class="c2"><span class="c3">1&micro;L DNA</span></p><p class="c2"><span class="c3">12&micro;L Master Mix</span></p><p class="c2"><span class="c3">0.5 &micro;L Phusion</span></p><p class="c2"><span class="c3">36.5&micro;L PCR Grade H</span><span class="c12">2</span><span class="c3">O</span></p><p class="c1"><span class="c3"></span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">Amplify with the following program:</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">98&deg;C for 30s</span></p><p class="c2"><span class="c3">35 Cycles of:</span></p><p class="c2"><span class="c3">98&deg;C for 10s</span></p><p class="c2"><span class="c3">47-65&deg;C for 30s</span></p><p class="c2"><span class="c3">72&deg;C for 30-45s/1kb</span></p><p class="c2"><span class="c3">72&deg;C for 10 minutes</span></p><p class="c2"><span class="c3">12&deg;C hold</span></p><p class="c1"><span class="c3"></span></p><p class="c2"><span class="c3">PCR Products were analyzed using the aforementioned gel electrophoresis protocol. The gel was loaded with 12uL of each sample and 10uL of ladder.</span></p><p class="c2"><span class="c3">&nbsp; </span></p><ol class="c14 lst-kix_28wz014s156k-0 start" start="1"><li class="c2 c4"><span class="c3">Ladder</span></li><li class="c1 c4"><span class="c3"></span></li><li class="c2 c4"><span class="c3">1:1 Dilution</span></li><li class="c1 c4"><span class="c3"></span></li><li class="c2 c4"><span class="c3">1:2 Dilution</span></li><li class="c1 c4"><span class="c3"></span></li><li class="c2 c4"><span class="c3">1:3 Dilution</span></li><li class="c1 c4"><span class="c3"></span></li></ol><p class="c2"><span class="c3">&nbsp;</span></p><p class="c2"><span class="c5">Transformation of Ligation Products</span></p><p class="c2"><span class="c13">The ligation products were transformed into Stellar cells at a volume of 20uL using the aforementioned transformation protocol. &nbsp;</span></p><p class="c1"><span class="c13"></span></p><p class="c1"><span></span></p>
   </div>

Difference between revisions of "Team:UConn/Notebook"

Revision as of 12:38, 4 November 2016

Notebook