Team:Aachen/Notebook/Wetlab/April

From 2014.igem.org

(Difference between revisions)
(→30th)
Line 1: Line 1:
__NOTOC__
__NOTOC__
{{Team:Aachen/Header}}
{{Team:Aachen/Header}}
 +
 +
 +
<html>
 +
<center>
 +
<ul class="menusmall-grid">
 +
<!-- Overview -->
 +
 +
  <li>
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Interlab_Study#isexperimentaldesign" style="color:black">
 +
    <div class="menusmall-item menusmall-info" ><div class="menukachel">Experi-mental Design</div></div>
 +
    <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/10/Aachen_14-10-13_Yellow_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
  <li>
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Interlab_Study#isexpectedresults" style="color:black">
 +
    <div class="menusmall-item menusmall-info" ><div class="menukachel">Expected Results</div></div>
 +
    <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/7/78/Aachen_14-10-13_Green_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
  <li>
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Interlab_Study#ismaterials" style="color:black">
 +
    <div class="menusmall-item menusmall-info" ><div class="menukachel">Materials & Methods</div></div>
 +
    <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/3/30/Aachen_14-10-13_Empty_Falcon_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
  <li>
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Interlab_Study#isresults" style="color:black">
 +
    <div class="menusmall-item menusmall-info" ><div class="menukachel">Results</div></div>
 +
    <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/b/b7/Aachen_14-10-13_Graph_Panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
<li>
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Interlab_Study#isdiscussion" style="color:black">
 +
    <div class="menusmall-item menusmall-info" ><div class="menukachel">Discussion</div></div>
 +
    <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/3/3b/Aachen_14-10-13_Graph_with_Error_Bars_Panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
</ul>
 +
</center>
 +
</html>
 +
<center>
<center>

Revision as of 10:12, 14 October 2014



previous month Wetlab main page next month

April

1st

Organization

where to get:

  • key cards and keys for the lab
  • cooled centifuge
  • space in -80 °C
  • bottles for 70 % ethanol
  • ... (problems of a first year team ^^)

12th

  • Chemically competent NEB Top10, DH5α and BL21 were made

15th

  • 800 ml LB for plates with 1.5% agar and kanamycin (50 µg/L) were prepared
  • The efficiency of our competent cells was tested
    • Colonies on the agar plates were counted after transformation with 1 µl DNA (147 ng/µl)
    • Cells had been incubated in SOC or LB medium
Dilution 200 µl (stock) 100 µl (stock) 1:5 1:10 1:100
DH5α SOC: 30 LB: 10 SOC: 7 LB: 1 SOC: 1 LB: 2 SOC: 2 LB: 1
NEB Top 10 SOC: 170 LB: 135 SOC: 100 LB: 79 SOC: 25 LB: 22 SOC: 9 LB: 9 SOC: 1 LB:0
  • BL21 cells were centrifuged and plated; only 128 colonies grew in total indicating a very bad efficiency (~482.11).

Following formula was used to calculate the efficiency:

Efficiency = (CFU /µg DNA) / Dilution
  • for DH5α: medial efficiency in SOC: 1864.36, and in LB: 440.68
  • for NEB: medial efficiency in SOC: 6779.66, and in LB: 4698.30
    Higher efficiency when using SOC!

22th

Pcq lab PCR advanced primus 25, 96

Lenght [bp]  % GC Tm TA
EYFP_RFC25 778 61 84 56
REACh1_C 481 62 84 57
REACh1_N 320 59 82 54
REACh2_C 487 62 83 57
REACh2_N 320 59 82 54

Program name IGEM.cyc

Parameter Duration Temp [°C]
Denature10:0098
Denature00:3098
Anneal00:3052
Elongate02:3672
Elongate05:0072
Storeforever8
  1. RFC25_F RFC25_R
  2. RFC25_F REACh1_R
  3. REACh1_F RFC25_R
  4. RFC25_F REACh2_R
  5. REACh2_F RFC25_R

23th

  • 5 µl of the PCR products were run on a 1,5% agarose gel
  • DNA bands were purified from gel with the High Pure Product Purification Kit. Full length products were contaminated with REACh1


  • SOE
  • ca. 30 ng/µL
20x -> 20 µL thereof: 30x
HF1010
dNTP44
Template2x 110
Phusion0.50.5
Primer-2x 2.5
Water33.521.5

Anneling: 52 °C

Elongation: 20 sec


  • PCR with Phusion polymerase
Volume [µL]
HF10
dNTP4
Template1
Phusion0.5
Primer2x 2.5
Water29.5
Total50
  • Digestion for restriction with NEB enzymes
Volume
Destination Vector500 ng
EcoRI-HF1 µL
PstI1 µL
10x NEB Buffer 2.15 µL
Water??? µL
Total??? µL
  • ligation with NEB Kit

28th

  • The DNA concentration of the SOE2 products were measured after purification
  • 1: 57 ng/µL
  • 2: 69.5 ng/µL

29th

  • PCR SOE1 and SOE2

30th

  • A PAGE of the SOE2 products was run on agarose gel for checking
    → no positive results; only full length products were amplified


  • SOE PCRs were run again
    → more template
    → hot start
    → faster
    → elongation: 15 min
    → 20 cycles
  • SOE1

Template A+B → 20 µL → 13.8 + 6.2 5.3 + 14.7

volume [µL]
HF10
dNTP4
Template20
Phusion0.5
Water13.5
  • SOE2


previous month Wetlab main page next month

Contact Disclaimer

Retrieved from "http://2014.igem.org/Team:Aachen/Notebook/Wetlab/April"