Difference between revisions of "Team:China Tongji/Notebook"
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<h5>June 6</h5> | <h5>June 6</h5> | ||
<p>1, Design the PCR primers of chR2-YFP.</p> | <p>1, Design the PCR primers of chR2-YFP.</p> | ||
+ | |||
<h4>1.1.1.2 Week2 -- June 8~14</h4> | <h4>1.1.1.2 Week2 -- June 8~14</h4> | ||
<h5>June 8</h5> | <h5>June 8</h5> | ||
− | <p>1,Theamplification of CHR2-YFP (use taq PCR protocol)</p> | + | <p>1, Theamplification of CHR2-YFP (use taq PCR protocol)</p> |
<p>2, AGE ( agarose gel electrophoresis )</p> | <p>2, AGE ( agarose gel electrophoresis )</p> | ||
<p>3, Gel extraction of chR2-YFP.</p> | <p>3, Gel extraction of chR2-YFP.</p> | ||
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<p>1, Select a single clone of each plate. (GFP, YFP, mcherry) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.</p> | <p>1, Select a single clone of each plate. (GFP, YFP, mcherry) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.</p> | ||
<p>2, Transformationof vector with pmyo-3(ppd95.77)</p> | <p>2, Transformationof vector with pmyo-3(ppd95.77)</p> | ||
− | + | <h5>June 10</h5> | |
+ | <p>1, Select a single clone of plate. (pmyo-3,ppd95.77) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.</p> | ||
+ | <p>2, Plasmid extraction ofE.coli DH5αwith GFP, YFP and mcherry in it.</p> | ||
+ | <h5>June 11</h5> | ||
+ | <p>1, Plasmid extraction of pmyo-3(ppd95.77)</p> | ||
+ | <p>2, Digestion of ppd95.77 with pmyo-3 in it and chR2-YFP using BamHI and EcoRI.(digestion protocol)</p> | ||
+ | <h5>June 12</h5> | ||
+ | <p>1, AGE ( agarose gel electrophoresis ) of digested vector---ppd95.77 with pmyo-3.</p> | ||
+ | <p>2, Gel extraction of pmyo-3.</p> | ||
+ | <p>3, purification of chR2-YFP witch has been digested with BamHI and EcoRI.</p> | ||
+ | <h5>June 14</h5> | ||
+ | <p>1, Transform of GFP, YFP, mcherryin E•coli BL21.</p> | ||
+ | |||
<h4>1.1.1.3 Week3 -- June 15~21</h4> | <h4>1.1.1.3 Week3 -- June 15~21</h4> | ||
− | <h4>1.1.1.4 Week4 -- June 22~25</h4> | + | <h5>June 15</h5> |
+ | <p>1, Ligation of pmyo-2 and chR2-YFP. (ligation protocol)</p> | ||
+ | <p>2, Transformation of ligation product: pmyo3-chR2-YFP (in ppd95.77).</p> | ||
+ | <h5>June 17</h5> | ||
+ | <p>1, Select a single clone of plate. (pmyo-3-chR2-YFP, ppd95.77) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.</p> | ||
+ | <p>2, Plasmid extraction of pmyo3-chR2-YFP.</p> | ||
+ | <h5>June 19</h5> | ||
+ | <p>1, Digestion of new plasmid: pmyo3-chR2-YFP using HindIII and EcoRI to check if the ligation step work or not. (it worked! Our first part is done!)</p> | ||
+ | <h5>June 20</h5> | ||
+ | <p>1, Transformation of pmyo3-chR2-YFP in order to get more plasmid.</p> | ||
+ | <p>2, Transformation of pmec3-chR2-YFP, pmec3-dsred and pmec4-chR2-YFP which are offered by professor Li’s lab.</p> | ||
+ | <p>3, Save the E.coli strain with glycerinum.</p> | ||
+ | <h5>June 21</h5> | ||
+ | <p>1, Theamplification of dsred and pmyo-2(use taq PCR protocol---to test the best temperature for the PCR).</p> | ||
+ | <p>2, the amplification of dsred and pmyo-2(use pfu PCR protocol).</p> | ||
+ | <p>3, AGE ( agarose gel electrophoresis ) of pmyo-2 and dsred.</p> | ||
+ | <p>4, Gel extraction of pmyo-2 and dsred.</p> | ||
+ | |||
+ | <h4>1.1.1.4 Week4 -- June 22~25</h4> | ||
+ | <h5>June 22</h5> | ||
+ | <p>1, digestion of pmyo3-chR2-YFP(using HindIII and BamHI)</p> | ||
+ | <p>2, AGE ( agarose gel electrophoresis ) of digested vector---ppd95.77 with chR2-YFP.</p> | ||
+ | <p>3, Gel extraction of pmyo-3(PPD95.77).</p> | ||
+ | <p>4, Digest of pmyo2 with HindIII and BamHI. Digest of dsred with BamHI and EcoRI. </p> | ||
+ | <p>5, gene purification for pmyo2 and dsred.</p> | ||
+ | <h5>June 24</h5> | ||
+ | <p>1, Ligation of pmyo2 with chR2-YFP (in ppd95.77).</p> | ||
+ | <p>2, Ligation of dsred with pmyo3 and pmyo2(in ppd95.77)</p> | ||
+ | <h5>June 25</h5> | ||
+ | <p>1, Digest of pmyo2-chR2-YFP and pmyo2-dsred and pmyo3-dsred.(usingHindIII and EcoRI) To check if we had ligated it successfully.</p> | ||
+ | <p>2, AGE ( agarose gel electrophoresis ) of digested products. Analyze the result.</p> | ||
</div> | </div> | ||
</div> | </div> | ||
+ | |||
<div class="fivePx"></div> | <div class="fivePx"></div> | ||
<div class="slidePanel slidePanel1 hoverHand"> | <div class="slidePanel slidePanel1 hoverHand"> | ||
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<div class="slideBlock" id="slideBlock1dot2"> | <div class="slideBlock" id="slideBlock1dot2"> | ||
<h4>1.1.2.1 Week2 -- July 11~14</h4> | <h4>1.1.2.1 Week2 -- July 11~14</h4> | ||
+ | <h5>July 11</h5> | ||
+ | <p>1, Design the PCR primers of Blink gene.</p> | ||
+ | <p>2, Design the PCR primers of pttx-3.</p> | ||
+ | <p>3, Design the PCR primers of ptrp4.</p> | ||
+ | <p>4, Prepare for competent cells</p> | ||
+ | <h5>July 14</h5> | ||
+ | <p>1, Transformation of the Blink plasmid which was kind offered by Anna’s lab.</p> | ||
+ | <p>2, Transformation of pmyo2-chR2-YFP, pmyo2-dsred and pmyo3-dsred in order to get more plasmids.</p> | ||
+ | <p>3, GFP, YFP, mcherry transform OP50 and PA14. (OP50 and PA14 are the food of C.elegans)</p> | ||
+ | |||
<h4>1.1.2.2 Week3 -- July 15~21</h4> | <h4>1.1.2.2 Week3 -- July 15~21</h4> | ||
+ | <h5>July 15</h5> | ||
+ | <p>1,Select single clones of plate. (pmyo-2-chR2-YFP, pmyo2-dsred and pmyo3-dsred ppd95.77) . Put the E.coli in 4ml LB buffer and cultivate for one night at 37℃.</p> | ||
+ | |||
+ | <h5>July 16</h5> | ||
+ | <p>1, Plasmid extraction of pmyo-2-chR2-YFP, pmyo2-dsred and pmyo3-dsred.</p> | ||
+ | |||
+ | <h5>July 17</h5> | ||
+ | <p>1, Recovery of </p> | ||
+ | <b>pNP260( Pnmr-1::flox::ChR2::mCherry.)</b> | ||
+ | <b>pCoS2(pnhr-79::Cre):</b> | ||
+ | <b>pCoS13(posm-10::loxP::LacZ::STOP::loxP:: ChR2::mCherry::SL2::GFP)</b> | ||
+ | <b>pSH116 (pdes-2::Cre)</b> | ||
+ | <p>which are kind offered by Alexander Gottschalk’ lab. </p> | ||
+ | <p>2, Transformation of pNP260, pCoS2, pCoS13 and pSH116.</p> | ||
+ | <p>3, Design of the PCR primers of pttx-HindIII-F and pttx3-Xbal-R.</p> | ||
+ | |||
+ | <h5>July 18</h5> | ||
+ | <p>1, Select single clones of plate.(pNP260, pCoS2, pCoS13 and pSH116). Put the E.coli in 4ml LB buffer and cultivate for 12h at 37℃.</p> | ||
+ | <p>2, Plasmid extraction.</p> | ||
+ | <p>3, Make genome DNA from worms.</p> | ||
+ | <p>4, Make LB plate and LB liquid.</p> | ||
+ | <p>5, Repeat the experiment: Prepare for competent cells: GFP, YFP, mcherry transform OP50 and PA14.</p> | ||
+ | <p>6, The amplification of Blink.</p> | ||
+ | <p>7, AGE ( agarose gel electrophoresis ) of blink PCR products. Analyze the result.</p> | ||
+ | <p>8, Gel extraction. (all at 300ng/ul)</p> | ||
+ | |||
+ | <h5>July 19</h5> | ||
+ | <p>1, Use the C.elegans genomic DNA as template and got pttx-3 with the help of Pttx3-HindIII-F and pttx-3-XbaI-R.</p> | ||
+ | <p>2, AGE ( agarose gel electrophoresis ) of PCR products. Analyze the result.</p> | ||
+ | |||
+ | <h5>July 20</h5> | ||
+ | <p>1, Transformation of P Blue plasmid.(used to make the mixture of microinjection liquid)</p> | ||
+ | <p>2, Select single clone on the culture plate.Put the E.coli in 4ml LB buffer and cultivate for 12h at 37℃.</p> | ||
+ | <p>3, Select a single clone of each plate and put each one in a tube that contain the ampicillin and 1ml LB culture media. (OP50 and PA14)</p> | ||
+ | <p>4, At 37℃ we culture them for 12h.</p> | ||
+ | |||
+ | <h5>July 21</h5> | ||
+ | <p>1, Add the mixture to a new 15ml tube and add in 4ml LB culture media.</p> | ||
+ | <p>2, Culture them for 3h until the OD number near 0.8.</p> | ||
+ | <p>3, Add 1mg IPTG in the liquid.</p> | ||
+ | <p>4, Culture them for 3h in order to gain the protein.</p> | ||
+ | |||
<h4>1.1.2.3 Week4 -- July 21~27</h4> | <h4>1.1.2.3 Week4 -- July 21~27</h4> | ||
+ | |||
<h4>1.1.2.4 Week5 -- July 28~31</h4> | <h4>1.1.2.4 Week5 -- July 28~31</h4> | ||
</div> | </div> |
Revision as of 18:58, 9 September 2015
Notebook
1. Record
- 1.1 Plasmid Part
- 1.2 Worm Part
- 1.3 Efficiency Part
- 1.4 Equipment Part
2. Timeline
1. Record
1.1 Plasmid Part
1.2 Worm Part
1.3 Efficiency Testpart
1.4 Equipment Part
2. Timeline
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