Team:ITB Indonesia/Notebook/WetLab

From 2013.igem.org

(Difference between revisions)
 
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<ul>
<ul>
   <li>Plasmid isolation until resuspension</li>
   <li>Plasmid isolation until resuspension</li>
 +
</ul>
 +
<p>Wednesday,  August 14th, 2013</p>
 +
<ul>
 +
  <li>Plasmid  isolation</li>
 +
  <li>Confirming  results from plasmid isolation by gel electrophoresis. Bands appeared between  3000-3500 bp. Predicted size for pSOS + GFP + pSBIK3 around 3274 bp and pUV +  GFP + pSBIK3 around 3158 bp excluding prefix and suffix</li>
 +
  <li>Predicted  size for PCR amplification: pSOS + GFP around 1070 bp and pUV + GFP around 954  bp excluding prefix and suffix</li>
 +
</ul>
 +
<p>Thursday,  August 15th, 2013</p>
 +
<ul>
 +
  <li>PCR  of pSOS + GFP + pSBIK3 and pUV + GFP + pSBIK3</li>
 +
  <li>Inoculating  E. coli BL21 (DE3) from glycerol stock to 5 mL of LB broth</li>
 +
</ul>
 +
<p>Friday,  August 16th of 2013</p>
 +
<ul>
 +
  <li>Gel  electrophoresis of PCR results from August 15th </li>
 +
</ul>
 +
<p>Saturday,  August 17th, 2013</p>
 +
<ul>
 +
  <li>Producing  competent <em>E. coli</em> BL21 (DE3) cells</li>
 +
  <li>Measuring  OD for E. coli cells in LB broth after 2 days of incubation</li>
 +
  <li>Scaling  up 500 uL of E. coli from LB culture to 50 mL of LB broth followed by OD  measurement and incubation until an OD of 0.35-0.45 was reached.</li>
 +
</ul>
 +
<p>Sunday,  August 18th, 2013</p>
 +
<ul>
 +
  <li>Making <em>E. coli</em> BL21 (DE3) glycerol stock</li>
 +
  <li>Transforming  pSOS + GFP + pSBIK3</li>
 +
  <li>Transforming  pUV + GFP + pSBIK3</li>
 +
</ul>
 +
<p>Monday,  August 19th, 2013</p>
 +
<ul>
 +
  <li>Growing  0.3 gram of freeze-dried E. coli</li>
 +
  <li>Incubation  result showed that 3 of 7 samples of LB cultures turned red</li>
 +
</ul>
 +
<p>Wednesday, August  21th, 2013</p>
 +
<ul>
 +
  <li>Restriction  digest of pUV</li>
 +
  <li>Ligation  of pUV + GFP + pSBIK3 followed by transformation into 50 uL of competent cells</li>
 +
</ul>
 +
<p>Thursday,  August 22nd, 2013</p>
 +
<ul>
 +
  <li>Results  from transformation in August 21st showed no colony growth</li>
 +
</ul>
 +
<p>Friday,  August 23rd, 2013</p>
 +
<ul>
 +
  <li>Ligation  of pUV + GFP + pSBIK3</li>
 +
</ul>
 +
<p>Saturday,  August 24th, 2013</p>
 +
<ul>
 +
  <li>Human  practice and fundraising at ITB '93 alumni meeting</li>
 +
  <li>Making  liquid culture of pUVGK1 and pUVGK2 using 5 mL of LB broth + 5 uL kanamycin</li>
 +
</ul>
 +
<p>Sunday,  August 25th, 2013</p>
 +
<ul>
 +
  <li>Plasmid  isolation of pUV + GFP + pSBIK3</li>
 +
  <li>PCR  of the results from plasmid isolation</li>
 +
  <li>Linearizing  pUV + GFP + pSBIK3 using X followed by gel eletrophoresis</li>
 +
  <li>Restriction  digest of linearized pUV + GFP + pSBIK3 using P</li>
 +
</ul>
 +
<p>Tuesday,  August 27th, 2013</p>
 +
<ul>
 +
  <li>Gel  electrophoresis of restriction product of pUV + GFP + pSBIK3. Results showed  that the plasmid was not successfully cut using P</li>
 +
  <li>Preparing  sample for DNA sequencing</li>
 +
  <li>Restriction  digest of linearized pUV + GFP + pSBIK3 using P (increased volume)</li>
 +
</ul>
 +
<p>Wednesday,  August 28 th, 2013</p>
 +
<ul>
 +
  <li>Gel  electrophoresis of restriction digest result from August 27th </li>
 +
  <li>Restriction  digest of pUV + GFP + pSBIK3 using X+P followed by gel electrophoresis. Results  showed that the digestion was failed.</li>
 +
  <li>Making  50 mL of PDA</li>
 +
  <li>Restriction  digest of pSOSGK5 using X+P</li>
 +
  <li>Ligating  the restriction digest result to pSBIC3 followed by transformation into liquid  culture</li>
 +
  <li>Restriction  digest of linearized pUV + GFP + pSBIK3 using P to check the enzyme performance.  The results showed that the enzyme performance was good.</li>
 +
  <li>Inoculating  pUVGK1 and pUVGK2 glycerol stock to 5 mL of LB broth</li>
 +
</ul>
 +
<p>Thursday,  August 29th, 2013</p>
 +
<ul>
 +
  <li>Ligating  pSOSGK5 to pSBIC3</li>
 +
  <li>Restriction  digest of linearized pUV + GFP using P</li>
 +
  <li>Restriction  digest of YFP using X+P</li>
 +
  <li>Ligating  pSOSG5 + PSBIC3 (pSOSGC5)</li>
 +
  <li>Ligating  pUVG1 + PSBIC3 (pUVGC1)</li>
 +
  <li>Ligating  pUVG2 + PSBIC3 (pUVGC2)</li>
 +
  <li>Ligating  pSOS + YFP + PSBIK3 (pSOSYK1)</li>
 +
  <li>Ligating  pUV + YFP + PSBIK3 (pUVYK2)</li>
 +
</ul>
 +
<p>Friday,  August 30th, 2013</p>
 +
<ul>
 +
  <li>Gel  electrophoresis of plasmid isolation results of pUV and BBa_J04450. The results  showed that both isolations were successful.</li>
 +
  <li>Restriction  digest of YFP and pSBIC3 using non-fast-digest X+P </li>
 +
  <li>Restriction  digest of pUVGK1, pUVGK2, and pUVGK3</li>
 +
</ul>
 +
<p>Monday,  September 2nd, 2013</p>
 +
<ul>
 +
  <li>Ligation  of pUV + YFP + pSBKI3</li>
 +
  <li>Ligation  of pSOS + YFP + pSBKI3</li>
 +
  <li>Transforming  pUVGC1, pUVGC2, pUVGC5</li>
 +
  <li>Transforming  pUV + YFP + pSBIK3 (pUVYK)</li>
 +
  <li>Transforming  pSOS + YFP + pSBIK3  (pSOSYK)</li>
 +
</ul>
 +
<p>Tuesday,  August 3rd, 2013</p>
 +
<ul>
 +
  <li>Making  LB broth</li>
 +
</ul>
 +
<p>Wednesday,  September 4th, 2013</p>
 +
<ul>
 +
  <li>Making  glycerol stock</li>
 +
  <li>Plasmid  isolation</li>
 +
  <li>Restriction  digest using E+S fast-digest</li>
 +
  <li>PCR  of the results from plasmid isolation followed by gel electrophoresis</li>
 +
  <li>Gel  electrophoresis of results from restriction digest</li>
 +
</ul>
 +
<p>Friday,  September 6th, 2013</p>
 +
<ul>
 +
  <li>Ligation  of pSOS + YFP + pSBIK3</li>
 +
  <li>Ligation  of pUV + YFP + pSBIK3</li>
 +
  <li>Transforming  the construct into E. coli DH5α</li>
 +
</ul>
 +
<p>Saturday, September  7th, 2013</p>
 +
<ul>
 +
  <li>Transforming  pUVGC2, pSOSGC5 into <em>E. coli</em> BL21  (DE3)</li>
 +
  <li>Repeating  the ligation of pSOS + YFP + pSBIK3 and pUV + YFP + pSBIK3</li>
 +
  <li>Ligating  pSOS + mCherry and pUV + mCherry</li>
 +
  <li>Restriction  digest of RBS using E+S and CP using E+S fast-digest</li>
 +
  <li>Restriction  digest of amyl CP and double terminator using X+P non-fast-digest</li>
 +
  <li>Confirming  the digestion results using gel electrophoresis</li>
 +
  <li>Ligation  of RBS + amyl CP K592009 + pSBIK3 followed by gel electrophoresis</li>
 +
</ul>
 +
<p>Sunday,  September 8th, 2013</p>
 +
<ul>
 +
  <li>Inocualting  pUVGC2 and pSOSGC5 to 14 mL of LB + chloramphenicol</li>
 +
  <li>Repeating  the restriction digest of amyl CP, mCherry, and double terminator using X+P  non-fast-digest</li>
 +
  <li>PCR  of digestion results of amyl CP followed by gel electrophoresis</li>
 +
  <li>Characterizing  pSOS</li>
 +
  <li>Purifying  PCR results of amyl CP</li>
 +
</ul>
 +
<p>Monday,  September 9th, 2013</p>
 +
<ul>
 +
  <li>Ligation  of pSOS + mCherry and pUV + mCherry</li>
 +
  <li>Gel  electrophoresis of purified PCR results of amyl CP</li>
 +
  <li>Restriction  digest of amyl CP using E+S fast-digest</li>
 +
  <li>Restriction  digest of amyl CP using X fast-digest + P non-fast-digest followed by gel  electrophoresis</li>
 +
  <li>Plasmid  isolation of 15E1</li>
 +
  <li>Ligation  of RBS (cut with E+S) + amyl CP (cut with X+P) + pSBIK3 (cut with E+P)</li>
 +
  <li>Ligation  of amyl CP (cut with E+S) + double terminator (cut with X+P) + pSBIK3 (cut with  E+P) followed by transformation</li>
 +
  <li>Making  freeze-dried <em>E. coli</em> cells</li>
 +
</ul>
 +
<p>Tuesday,  September 10th, 2013</p>
 +
<ul>
 +
  <li>Ligation  of pUV + mCherry and pSOS + mCherry</li>
 +
  <li>Checking  growth of pSOS + mCherry on LB broth + Kanamycin</li>
 +
</ul>
 +
<p>Wednesday,  September 11th, 2013</p>
 +
<ul>
 +
  <li>Transforming  pSOS + mCherry</li>
 +
  <li>Transforming  pUV + mCherry</li>
 +
  <li>Restriction  digest of scale-up mCherry using X fast-digest + P non-fast-digest</li>
 +
</ul>
 +
<p>Thursday,  September 12th, 2013</p>
 +
<ul>
 +
  <li>Plasmid  isolation of 4B2 stock culture</li>
 +
  <li>Measuring  OD for freeze-dried <em>E. coli</em> cells</li>
 +
  <li>Restriction  digest of 4B2 using X+S</li>
 +
</ul>
 +
<p>Friday,  September 13th, 2013</p>
 +
<ul>
 +
  <li>Restriction  digest of 4B2 using X+S followed by gel electrophoresis and purification</li>
 +
  <li>Mearusing  OD for freeze-dried <em>E. coli</em> cells</li>
 +
  <li>Making  freeze-dried <em>E. coli</em> cells</li>
 +
  <li>Plasmid  isolation of 4B2 and pSOS + mCherry + pSBIK3 followed by gel electrophoresis</li>
 +
</ul>
 +
<p>Saturday,  September 14th, 2013</p>
 +
<ul>
 +
  <li>Checking  pSOS + mCherry + pSBIK3 by PCR and restriction digest using E+S followed by gel  electrophoresis</li>
 +
  <li>Repeating  ligation of pSOS + mCherry + pSBIK3 and pUV + mCherry + pSBIK3</li>
 +
  <li>Inoculating  pSOS + mCherry + pSBIK3 glycerol stock to 5 mL of LB broth + 5 uL of kanamycin</li>
 +
</ul>
 +
<p>Sunday,  September 15th, 2013</p>
 +
<ul>
 +
  <li>Transforming</li>
 +
  <li>Repeating  the restriction digest of 4B2 followed by gel electrophoresis</li>
 +
  <li>Inoculating  pSOS + mCherry + pSBIK3 stock culture to 5 mL of LB broth + 5 uL kanamycin</li>
 +
  <li>Inoculating  pUV + mCherry + pSBIK3 stock culture to 5 mL of LB broth + 5 uL kanamycin</li>
 +
</ul>
 +
<p>Monday,  September 16th, 2013</p>
 +
<ul>
 +
  <li>Making  glycerol stock of pUV + mCherry + pSBIK3 and pSOS + mCherry + pSBIK3</li>
 +
  <li>Gel  electrophoresis of pUVmCherryK3 and pSOSmCherryK3</li>
 +
</ul>
 +
<p>Tuesday,  September 17th, 2013</p>
 +
<ul>
 +
  <li>Plasmid  isolation of CYP 1-9, mCherry, and pSOS followed by gel electrophoresis</li>
 +
  <li>Making  new construct followed by restriction digest</li>
 +
  <li>Inoculating  E. coli DH5α cells from glycerol stock to 3 mL of LB broth for making competent  cells</li>
 +
  <li>Making  SDS PAGE extraction solution, SDS 10% stock solution, and Na3PO4  1 M stock solution</li>
 +
</ul>
 +
<p>Wednesday,  September 18th, 2013</p>
 +
<ul>
 +
  <li>Making  competent <em>E. coli</em> DH5α cells and  pSOS/pUV liquid culture</li>
 +
  <li>Ligation</li>
 +
</ul>
 +
<p>Thursday,  September 19th, 2013</p>
 +
<ul>
 +
  <li>Discussion  with advisors</li>
 +
  <li>Transforming  CYP 1, 2, 3</li>
 +
  <li>Restriction  digest of pSBIK3 using P non-fast-digest + E fast-digest</li>
 +
  <li>Ligation  of pSBIK3 + pSOS + mCherry and pUV + mCherry + pSBIK3</li>
 +
  <li>Restriction  digest of CYP8 followed by ligation</li>
 +
</ul>
 +
<p>Friday,  September 20th, 2013</p>
 +
<ul>
 +
  <li>Measuring  OD for freeze-dried <em>E. coli</em> cells</li>
 +
</ul>
 +
<p>Saturday,  September 21st, 2013</p>
 +
<ul>
 +
  <li>Transforming  ligation product of CYP 1, 2, 3 into <em>E.  coli</em> DH5α cells</li>
 +
  <li>PCR  of CYP 1-9 followed by gel lelectrophoresis</li>
 +
  <li>Plasmid  isolation of pUV + mCherry + pSBIK3 followed by gel electrophoresis</li>
 +
</ul>
 +
<p>Sunday, September  22nd, 2013</p>
 +
<ul>
 +
  <li>Repeating  ligation of pSOS + mCherry + pSBIK3 and pUV + mCHerry + pSBIK3 followed by  transformation into E. coli DH5α cells</li>
 +
  <li>Restriction  digest of CYP 2 using E, E+X, and E+P</li>
 +
  <li>Restriction  digest of CYP 9 using E+P</li>
 +
  <li>Restriction  digest of pUV +mCherry using E</li>
 +
  <li>PCR  of CYP 2 and pUV + mCherry followed by gel electrophoresis</li>
 +
  <li>Restriction  digest of pSBIK3 using E+P and E+P non-fast-digest</li>
 +
  <li>Restriction  digest of CYP 2 and CYP 9 using E+P non-fast-digest followed by gel  electrophoresis</li>
 +
</ul>
 +
<p>Monday, September  23rd, 2013</p>
 +
<ul>
 +
  <li>Plasmid  isolation of CYP 2 and CYP 9 followed by gel electrophoresis, gel purification,  and restriction digest using E+P</li>
 +
  <li>Measuring  OD for 0.85 g of freeze-dried E. coli cells in 25 mL of LB broth + 5 mL of  glucose 10%</li>
 +
  <li>Sampling  of colonies containing pSOS/pUV + mCherry</li>
 +
</ul>
 +
<p>Tuesday,  September 24th, 2013</p>
 +
<ul>
 +
  <li>Plasmid  isolation of CYP from 12 samples</li>
 +
  <li>Restriction  digest of plasmid isolation results using E fast-digest</li>
 +
  <li>PCR  of CYP followed by gel electrophoresis</li>
 +
  <li>Plasmid  isolation of pUV + mCherry + pSBIK3 and pSOS + mCherry + pSBIK3 followed by gel  electrophoresis</li>
 +
  <li>Restriction  digest of CYP A-L (12 samples) </li>
 +
  <li>Scaling  up of pUV + GFP + pSBIC3 and pSOS + GFP + pSBIC3 glycerol stock to 5 mL of LB  broth + 5 uL chloramphenicol and then continued to 50 mL of LB broth + 50 uL  chloramphenicol</li>
 +
  <li>GFP  expression test for pUVGC1 and pSOSGC1 using UV light and fluorescence  microscope</li>
 +
  <li>Gel  electrophoresis of pUV1, pUV2, pSOS1, pSOS2, pSOS3, and pSOS4</li>
 +
</ul>
 +
<p>Wednesday,  September 25th, 2013</p>
 +
<ul>
 +
  <li>Co-transforming  pUVGK2, pSOSGK5, and CYP 9 into <em>E. coli</em> DE3 (BL21) cells</li>
 +
  <li>Measuring  OD for pUV and pSOS stock cultures</li>
 +
  <li>Restriction  digest using XbaI and Xba  +SpeI </li>
 +
  <li>Gel  electrophoresis</li>
 +
</ul>
 +
<p>Thursday, September 26th, 2013</p>
 +
<ul>
 +
  <li>Extracting Aflatoxin from <em>Aspergillus flavus</em> using chloroform, and checking whether our  extraction succesful or not by looking under UV light. Our extract glowing  yellow-green!</li>
 +
</ul>
 +
<p>Friday, September 27th, 2013</p>
 +
<ul>
 +
  <li>Testing our construct with Aflatoxin</li>
 +
  <li>Finishing our WIKI</li>
</ul>
</ul>
</div>
</div>

Latest revision as of 03:05, 28 September 2013

WetLab Notebook

June 2013

Saturday, 1 June 2013
Making CCMB 80 buffer and CaCl2 + Gliserin 15% buffer.

Sunday, 2 June 2013
Making competent cell with iGEM standard method (CCMB 80 Buffer) and with CaCl2 + Gliserin 15% buffer.

Tuesday, 4 June 2013
Transform the plasmid to DH5α competent cell. We compared number of colony from CaCl2 method and  from iGEM standard method. For each method, we spread 20 µl and 200 µl culture.

Wednesday, 5 June 2013
Count the transform colony. Wehave found that the number of colony from 20 µl with CCMB 80 Buffer gave  a better result than 200 µl with CaCl2 + Gliserin 15% buffer. So, we are confident with iGEM standard method.

Thursday, 6 June 2013
We try varying the time and repetition of heat shock during transformation. We use single heat shock and double heat shock, and 60 sec and 90 sec for each method.

Friday, 7 June 2013
Count the transformation colony. Wehave found that the number of colony from single heat shock for 60 sec giving the best result than other. So, we applied this method for our transformation.

Saturday, 8 June 2013
Repeating transformation like a day before

Monday, 10 June 2013

  • Transform xxx using competent cell and agar plate made of SOB, heat shock 60 seconds 42⁰C, duplo.

Tuesday, 11 june 2013

  • Making SOC medium
  • Transforming BBa_J04450 (pSB1C3) from transformation eficiency kit (concentration 5pg/ µl) with 1M IPTG induction : 0 µl, 2 µl, 4 µl
  • Making 3ml DH5α  broth culture from gliserol stock
  • Counting the transformacy efficiency from the day before. From first plate resulting 300 colony, and 258 colony from the second
  • Discussing 3A assembly system and white screening selection
  • Making LB agar stock

Thursday, 13 June 2013

  • Making gliserol stock of BBa_J000450. The colonies grew
  • Making glycerol stock of BBa_E0430. The colonies grew well.

Friday, June 14th 2013

  • Transforming BBa_J22106. The colonies grew poorly
  • Transforming BBa_J06702. The colonies grew well.
  • Transforming BBa_I13507. The colonies grew well.
  • Transforming BBa_I765001. The colonies grew poorly.
  • Transforming BBa_E0840. The colonies grew well.

Friday, June 14th 2013

  • Preparing solutions for plasmid isolation by alkali lysis method
  • Making glycerol stock of 4B2 and 20K3
  • Making liquid culture of 9N5, 16B3, 23C3

Monday, June 17th, 2013

  • BBa_K592009
  • BBa_E1010
  • BBa_K592011
  • BBa_K592010

Tuesday, June 18th, 2013

  • Making liquid culture of 19E1 and 12N3
  • Transforming BBa_B0015
  • Transforming BBa_B1006
  • Making 5X ligation buffer

Wednesday, June 19th, 2013

  • Making stock culture of 19E1 and 12N3
  • Plasmid isolation from 19E1 and 12N3 stock culture
  • Making 1M Tris solution, pH 7.5
  • Making 0.5M EDTA slution, pH 8.0

Thursday, June 20th, 2013

  • Making stock culture of 4F3 and 6D3
  • Plasmid isolation from 4F3 and 6D3 stock culture
  • Making and sterilizing TE Buffer

Friday, June 21st, 2013

  • Transforming BBa_K873002
  • Trannsforming BBa_B0034

Saturday, June 22nd, 2013

  • Making glycerol stock of 3H1
  • Plasmid isolation from 3H1 stock culture
  • Discussing the planned system

Sunday, 23rd, 2013

  • Transforming BBa_J23119

Monday, June 24th, 2013

  • Confirming results from plasmid isolation by gel electrophoresis

Tuesday, June 25th, 2013

  • Plasmid isolation from 18D3 and 18A5 stock culture

Thursday, June 27th, 2013

  • Plasmid isolation from 20K3, 16B3, 23C3, 21B5, 9N5, 19E1, 12N3, 4F3, 6D3, and 3HI stock culture
  • Confirming results from plasmid isolation in June 25 and 27th by gel electrophoresis

Sunday, June 30th, 2013

  • Inoculating (parts?) in 25mL of LB broth + 25 uL of antibiotic

July 2013

Monday, July 1st, 2013

  • Transforming RBS 1 (strong) B0030. The colonies grew well.
  • Gel electrophoresis of 16B3, 23C3, 21B5, 9N5
  • Inoculating 4F3, 6D3, 3H1, 18D3, and 18A5 in 25mL of LB broth + 25 uL of antibiotic

Tuesday, July 2nd, 2013

  • Plasmid isolation from 4F3, 6D3, 3H1, 18D3, and 18A5 stock culture using alkali-lysis buffer solution
  • Making liquid culture from colonies in agar plate (?)
  • Preparing solutons for restriction digest (1X NE Buffer 1, 1X NE Buffer 2, 1X NE Buffer 3, 1X NE Buffer 4)
  • Confirming results from plasmid isolation by gel electrophoresis
  • Restriction digest of 9N5 (Part A, SOS Promoter) and 23C3 (Part B, GFP Generator) and assembly using 3A method

Wedenesday, July 3rd, 2013

  • Making NaCl 5M stock solution
  • Gel purification of 4F3 and 3H1
  • Restriction digest of 16B3 using E+P
  • Confirming digestion results by gel electrophoresis. The resulting band was so thin.
  • Restriction digest of 16B3 using E+X and 21B5 using E+S

Monday, July 8th 2013

  • Restriction digest  of 4B2 using E+P, 23C3 using X+P, 21B5 using E+S, 9N5 using E+S.
  • Assembling the digestion product using 3A method

Thursday, July 11th, 2013

  • Plasmid isolation of pSBIK3
  • Confirming isolation results by gel electrophoresis
  • Restriction digest of plasmid backbone pSBIK3
  • Ligation of SOS Promoter + GFP + pSBIK3
  • Transforming ligation result into 50 uL of competent cells
  • Confirming result from plasmid isolation (1G5, 2H2, 5A5, 6B2) by gel electrophoresis. No band appeared.

Saturday, July 13rd, 2013

  • Inoculating pUV + GFP to LB broth
  • Making stock culture of pSOS + GFP. Centrifugation result showed that the pSOS + GFP failed to insert into the backbone, hence the transformation, inoculation, and isolation steps was repeated.
  • Ligation of pUV + GFP + pSBKI3 in 4oC overnight

Sunday, July 14th, 2013

  • Centrifugation result showed that the pUV + GFP sucessfully inserted into the backbone
  • Plasmid isolation of 2.1, 2.2, 2.3, and 2.4, stored in -20oC
  • Confirming result from plasmid isolation by gel electrophoresis
  • Amplified the result by PCR

Monday, July 15th, 2013

  • Restriction digest of plasmid 2.3 using EcoRI and Pst I
  • Confirming result from restriction digest by gel electrophoresis. The result showed that the digestion was failed.
  • Plasmid isolation from pSOS + GFP + pSBIK3
  • Preparing 200 mL of LB agar

Tuesday, July 16th, 2013

  • Scaling up the pSBKI3 stock culture
  • Confirming result from plasmid isolation of pSOSGK1, pSOSGK2, pSOSGK3, pSOSGK4, pSOSGK5 by gel electrophoresis.

Wednesday, July 17th, 2013

  • Repeating the restriction digest using E+P
  • Plasmid isolation from scale up culture of pSBKI3, pSBIA3

Thursday, July 18th, 2013

  • Preparing 5 mL of liquid broth containing E. coli  BL21 (DE3)
  • Plasmid isolation of 2.2 from stock culture
  • Confirming result from plasmid isolation of 2.2 and restriction digest for pUV, 23C3, 9N5, 6B2 by gel electrophoresis. The result was failed.

Friday, July 19th, 2013

  • Preparing 50 mL of liquid brith containing E. coli BL21 (DE3)
  • Resuspending the culture in 70% ethanol
  • Optimizing condition for restriction digest (4oC for 14-16 h vs 37oC for 4 h) using GFP, X, and P

Saturday, July 20th, 2013

  • Ending the overight incubation
  • PCR of 2.1, 2.3, and 2.4 (results from plasmid isoaltion of pUV+GFP) using VF2 and V2 primers.
  • Gel electrophoresis of 2.1, 2.2, and 2.4. The result was failed possibly due to high annealing temperature (64oC).
  • Repeating PCR of 2.1, 2.3, and 2.4 using lower temperatures (60oC) followed by gel electrophoresis. The result was successful.
  • PCR of pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5 followed by electrophoresis. The result was successful

Monday, July 22nd, 2013

  • Restriction digest of 23C3, 2.1, 2.3, and 2.4 using X+P and buffer H
  • Scaling up of 23C3 from glycerol stock
  • Gel electrophoresis of 23C3, 2.1, 2.3, and 2.4
  • Scaling up of pUV + GFP + pSBKI3 from glycerol stock

Tuesday, July 23rd, 2013

  • Plasmid isolation of 23C3
  • Scaling up of pUV + GFP
  • Restriction digest of pSOS + GK1 using X+P
  • Gel electrophoresis of pSOSGK4 uncut, 23C3, and scaled-up pUV + GFP

Thursday, July 25th, 2013

  • Restriction digest of monomeric RFP (mRFP) BBa_E1010
  • Ligation of pSOS + GFP + pSBIK3
  • Transforming restriction and ligation results
  • PCR of pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5 followed by gel electrophoresis. The result was successful

Friday, July 26th, 2013

  • Double restriction digest of pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5, and pSBIK3 using X and then using P
  • Making liquid culture of pSOS + GFP + pSBKI3, incubated for 18 h at 37oC.
  • Gel electrophoresis of pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5. The result was successful

Saturday, July 27th, 2013

  • Gel electrophoresis of digestion results using X+P
  • Gel electrophoresis of isolated pSOS + GFP

Sunday, July 28th, 2013

  • Project presentation to Mrs. Betty

Monday, July 29th, 2013

  • Repeating the restriction digest of pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5, and pSBIK3
  • Restriction digest of pSBIC3 plasmid backbone, incubated for 4 h at 37oC
  • Ligation of pSBIC3 + pUV + GFP and pSBIC3 + pSOSGK5
  • Repeating the gel electrophoresis for pSOSGK1, pSOSGK 2, pSOSGK3, pSOSGK4, pSOSGK5, and pSBIK3, including digestion result for pSBIC3
  • Transforming the ligation results. The colonies didn’t grow.

Tuesday, July 30th, 2013

  • Repeating the restriction digest of pSBIC3 from 12N3
  • Restriction digest of pSBIC3 from BBa_J04450
  • PCR of pSOS + GFP + pSBIK3 followed by electrophoresis
  • Inoculating BBa_J04450 from glycerol stock to 25 mL of liquid broth
  • Discussing about the next step of making dried E. coli cell
  • Making 250 mL of LB broth

Wednesday, July 31st, 2013

  • Measuring growth rate and plotting growth curve for dried E. coli cell

August 2013

Thursday, August 1st, 2013

  • Preparing solutions for plasmid isolation
  • Making 250 mL of LB agar and 250 mL of LB broth
  • Inoculating colonies of pSOS + GFP + pSBIK3
  • Preparation for DNA sequencing

Friday, August 2nd, 2013

  • Sending DNA sequence (2.4, pSOGK1, pSOGK5) to Macrogen
  • Observing results from inoculation
  • Preparing 5 mL of LB broth

Saturday, August 3rd, 2013

  • Plasmid isolation until resuspension

Wednesday, August 14th, 2013

  • Plasmid isolation
  • Confirming results from plasmid isolation by gel electrophoresis. Bands appeared between 3000-3500 bp. Predicted size for pSOS + GFP + pSBIK3 around 3274 bp and pUV + GFP + pSBIK3 around 3158 bp excluding prefix and suffix
  • Predicted size for PCR amplification: pSOS + GFP around 1070 bp and pUV + GFP around 954 bp excluding prefix and suffix

Thursday, August 15th, 2013

  • PCR of pSOS + GFP + pSBIK3 and pUV + GFP + pSBIK3
  • Inoculating E. coli BL21 (DE3) from glycerol stock to 5 mL of LB broth

Friday, August 16th of 2013

  • Gel electrophoresis of PCR results from August 15th

Saturday, August 17th, 2013

  • Producing competent E. coli BL21 (DE3) cells
  • Measuring OD for E. coli cells in LB broth after 2 days of incubation
  • Scaling up 500 uL of E. coli from LB culture to 50 mL of LB broth followed by OD measurement and incubation until an OD of 0.35-0.45 was reached.

Sunday, August 18th, 2013

  • Making E. coli BL21 (DE3) glycerol stock
  • Transforming pSOS + GFP + pSBIK3
  • Transforming pUV + GFP + pSBIK3

Monday, August 19th, 2013

  • Growing 0.3 gram of freeze-dried E. coli
  • Incubation result showed that 3 of 7 samples of LB cultures turned red

Wednesday, August 21th, 2013

  • Restriction digest of pUV
  • Ligation of pUV + GFP + pSBIK3 followed by transformation into 50 uL of competent cells

Thursday, August 22nd, 2013

  • Results from transformation in August 21st showed no colony growth

Friday, August 23rd, 2013

  • Ligation of pUV + GFP + pSBIK3

Saturday, August 24th, 2013

  • Human practice and fundraising at ITB '93 alumni meeting
  • Making liquid culture of pUVGK1 and pUVGK2 using 5 mL of LB broth + 5 uL kanamycin

Sunday, August 25th, 2013

  • Plasmid isolation of pUV + GFP + pSBIK3
  • PCR of the results from plasmid isolation
  • Linearizing pUV + GFP + pSBIK3 using X followed by gel eletrophoresis
  • Restriction digest of linearized pUV + GFP + pSBIK3 using P

Tuesday, August 27th, 2013

  • Gel electrophoresis of restriction product of pUV + GFP + pSBIK3. Results showed that the plasmid was not successfully cut using P
  • Preparing sample for DNA sequencing
  • Restriction digest of linearized pUV + GFP + pSBIK3 using P (increased volume)

Wednesday, August 28 th, 2013

  • Gel electrophoresis of restriction digest result from August 27th
  • Restriction digest of pUV + GFP + pSBIK3 using X+P followed by gel electrophoresis. Results showed that the digestion was failed.
  • Making 50 mL of PDA
  • Restriction digest of pSOSGK5 using X+P
  • Ligating the restriction digest result to pSBIC3 followed by transformation into liquid culture
  • Restriction digest of linearized pUV + GFP + pSBIK3 using P to check the enzyme performance. The results showed that the enzyme performance was good.
  • Inoculating pUVGK1 and pUVGK2 glycerol stock to 5 mL of LB broth

Thursday, August 29th, 2013

  • Ligating pSOSGK5 to pSBIC3
  • Restriction digest of linearized pUV + GFP using P
  • Restriction digest of YFP using X+P
  • Ligating pSOSG5 + PSBIC3 (pSOSGC5)
  • Ligating pUVG1 + PSBIC3 (pUVGC1)
  • Ligating pUVG2 + PSBIC3 (pUVGC2)
  • Ligating pSOS + YFP + PSBIK3 (pSOSYK1)
  • Ligating pUV + YFP + PSBIK3 (pUVYK2)

Friday, August 30th, 2013

  • Gel electrophoresis of plasmid isolation results of pUV and BBa_J04450. The results showed that both isolations were successful.
  • Restriction digest of YFP and pSBIC3 using non-fast-digest X+P
  • Restriction digest of pUVGK1, pUVGK2, and pUVGK3

Monday, September 2nd, 2013

  • Ligation of pUV + YFP + pSBKI3
  • Ligation of pSOS + YFP + pSBKI3
  • Transforming pUVGC1, pUVGC2, pUVGC5
  • Transforming pUV + YFP + pSBIK3 (pUVYK)
  • Transforming pSOS + YFP + pSBIK3  (pSOSYK)

Tuesday, August 3rd, 2013

  • Making LB broth

Wednesday, September 4th, 2013

  • Making glycerol stock
  • Plasmid isolation
  • Restriction digest using E+S fast-digest
  • PCR of the results from plasmid isolation followed by gel electrophoresis
  • Gel electrophoresis of results from restriction digest

Friday, September 6th, 2013

  • Ligation of pSOS + YFP + pSBIK3
  • Ligation of pUV + YFP + pSBIK3
  • Transforming the construct into E. coli DH5α

Saturday, September 7th, 2013

  • Transforming pUVGC2, pSOSGC5 into E. coli BL21 (DE3)
  • Repeating the ligation of pSOS + YFP + pSBIK3 and pUV + YFP + pSBIK3
  • Ligating pSOS + mCherry and pUV + mCherry
  • Restriction digest of RBS using E+S and CP using E+S fast-digest
  • Restriction digest of amyl CP and double terminator using X+P non-fast-digest
  • Confirming the digestion results using gel electrophoresis
  • Ligation of RBS + amyl CP K592009 + pSBIK3 followed by gel electrophoresis

Sunday, September 8th, 2013

  • Inocualting pUVGC2 and pSOSGC5 to 14 mL of LB + chloramphenicol
  • Repeating the restriction digest of amyl CP, mCherry, and double terminator using X+P non-fast-digest
  • PCR of digestion results of amyl CP followed by gel electrophoresis
  • Characterizing pSOS
  • Purifying PCR results of amyl CP

Monday, September 9th, 2013

  • Ligation of pSOS + mCherry and pUV + mCherry
  • Gel electrophoresis of purified PCR results of amyl CP
  • Restriction digest of amyl CP using E+S fast-digest
  • Restriction digest of amyl CP using X fast-digest + P non-fast-digest followed by gel electrophoresis
  • Plasmid isolation of 15E1
  • Ligation of RBS (cut with E+S) + amyl CP (cut with X+P) + pSBIK3 (cut with E+P)
  • Ligation of amyl CP (cut with E+S) + double terminator (cut with X+P) + pSBIK3 (cut with E+P) followed by transformation
  • Making freeze-dried E. coli cells

Tuesday, September 10th, 2013

  • Ligation of pUV + mCherry and pSOS + mCherry
  • Checking growth of pSOS + mCherry on LB broth + Kanamycin

Wednesday, September 11th, 2013

  • Transforming pSOS + mCherry
  • Transforming pUV + mCherry
  • Restriction digest of scale-up mCherry using X fast-digest + P non-fast-digest

Thursday, September 12th, 2013

  • Plasmid isolation of 4B2 stock culture
  • Measuring OD for freeze-dried E. coli cells
  • Restriction digest of 4B2 using X+S

Friday, September 13th, 2013

  • Restriction digest of 4B2 using X+S followed by gel electrophoresis and purification
  • Mearusing OD for freeze-dried E. coli cells
  • Making freeze-dried E. coli cells
  • Plasmid isolation of 4B2 and pSOS + mCherry + pSBIK3 followed by gel electrophoresis

Saturday, September 14th, 2013

  • Checking pSOS + mCherry + pSBIK3 by PCR and restriction digest using E+S followed by gel electrophoresis
  • Repeating ligation of pSOS + mCherry + pSBIK3 and pUV + mCherry + pSBIK3
  • Inoculating pSOS + mCherry + pSBIK3 glycerol stock to 5 mL of LB broth + 5 uL of kanamycin

Sunday, September 15th, 2013

  • Transforming
  • Repeating the restriction digest of 4B2 followed by gel electrophoresis
  • Inoculating pSOS + mCherry + pSBIK3 stock culture to 5 mL of LB broth + 5 uL kanamycin
  • Inoculating pUV + mCherry + pSBIK3 stock culture to 5 mL of LB broth + 5 uL kanamycin

Monday, September 16th, 2013

  • Making glycerol stock of pUV + mCherry + pSBIK3 and pSOS + mCherry + pSBIK3
  • Gel electrophoresis of pUVmCherryK3 and pSOSmCherryK3

Tuesday, September 17th, 2013

  • Plasmid isolation of CYP 1-9, mCherry, and pSOS followed by gel electrophoresis
  • Making new construct followed by restriction digest
  • Inoculating E. coli DH5α cells from glycerol stock to 3 mL of LB broth for making competent cells
  • Making SDS PAGE extraction solution, SDS 10% stock solution, and Na3PO4 1 M stock solution

Wednesday, September 18th, 2013

  • Making competent E. coli DH5α cells and pSOS/pUV liquid culture
  • Ligation

Thursday, September 19th, 2013

  • Discussion with advisors
  • Transforming CYP 1, 2, 3
  • Restriction digest of pSBIK3 using P non-fast-digest + E fast-digest
  • Ligation of pSBIK3 + pSOS + mCherry and pUV + mCherry + pSBIK3
  • Restriction digest of CYP8 followed by ligation

Friday, September 20th, 2013

  • Measuring OD for freeze-dried E. coli cells

Saturday, September 21st, 2013

  • Transforming ligation product of CYP 1, 2, 3 into E. coli DH5α cells
  • PCR of CYP 1-9 followed by gel lelectrophoresis
  • Plasmid isolation of pUV + mCherry + pSBIK3 followed by gel electrophoresis

Sunday, September 22nd, 2013

  • Repeating ligation of pSOS + mCherry + pSBIK3 and pUV + mCHerry + pSBIK3 followed by transformation into E. coli DH5α cells
  • Restriction digest of CYP 2 using E, E+X, and E+P
  • Restriction digest of CYP 9 using E+P
  • Restriction digest of pUV +mCherry using E
  • PCR of CYP 2 and pUV + mCherry followed by gel electrophoresis
  • Restriction digest of pSBIK3 using E+P and E+P non-fast-digest
  • Restriction digest of CYP 2 and CYP 9 using E+P non-fast-digest followed by gel electrophoresis

Monday, September 23rd, 2013

  • Plasmid isolation of CYP 2 and CYP 9 followed by gel electrophoresis, gel purification, and restriction digest using E+P
  • Measuring OD for 0.85 g of freeze-dried E. coli cells in 25 mL of LB broth + 5 mL of glucose 10%
  • Sampling of colonies containing pSOS/pUV + mCherry

Tuesday, September 24th, 2013

  • Plasmid isolation of CYP from 12 samples
  • Restriction digest of plasmid isolation results using E fast-digest
  • PCR of CYP followed by gel electrophoresis
  • Plasmid isolation of pUV + mCherry + pSBIK3 and pSOS + mCherry + pSBIK3 followed by gel electrophoresis
  • Restriction digest of CYP A-L (12 samples)
  • Scaling up of pUV + GFP + pSBIC3 and pSOS + GFP + pSBIC3 glycerol stock to 5 mL of LB broth + 5 uL chloramphenicol and then continued to 50 mL of LB broth + 50 uL chloramphenicol
  • GFP expression test for pUVGC1 and pSOSGC1 using UV light and fluorescence microscope
  • Gel electrophoresis of pUV1, pUV2, pSOS1, pSOS2, pSOS3, and pSOS4

Wednesday, September 25th, 2013

  • Co-transforming pUVGK2, pSOSGK5, and CYP 9 into E. coli DE3 (BL21) cells
  • Measuring OD for pUV and pSOS stock cultures
  • Restriction digest using XbaI and Xba +SpeI
  • Gel electrophoresis

Thursday, September 26th, 2013

  • Extracting Aflatoxin from Aspergillus flavus using chloroform, and checking whether our extraction succesful or not by looking under UV light. Our extract glowing yellow-green!

Friday, September 27th, 2013

  • Testing our construct with Aflatoxin
  • Finishing our WIKI

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