Team:Aachen/Notebook/Wetlab/April

From 2014.igem.org

(Difference between revisions)
Line 10: Line 10:
   <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
   <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/March" style="color:black">
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/March" style="color:black">
-
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel">Go to March</div></div>
+
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to March</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/7/7a/Aachen_14-10-10_March_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/7/7a/Aachen_14-10-10_March_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     </div>
     </div>
Line 18: Line 18:
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/April" style="color:black">
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/April" style="color:black">
-
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel">Go to April</div></div>
+
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to April</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/2/2d/Aachen_14-10-10_April_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/2/2d/Aachen_14-10-10_April_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     </div>
     </div>
Line 26: Line 26:
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/May" style="color:black">
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/May" style="color:black">
-
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel">Go to May</div></div>
+
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to May</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/6/67/Aachen_14-10-10_May_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/6/67/Aachen_14-10-10_May_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     </div>
     </div>
Line 34: Line 34:
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/June" style="color:black">
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/June" style="color:black">
-
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel">Go to June</div></div>
+
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to June</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/1d/Aachen_14-10-10_June_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/1d/Aachen_14-10-10_June_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     </div>
     </div>
Line 42: Line 42:
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/July" style="color:black">
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/July" style="color:black">
-
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel">Go to July</div></div>
+
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to July</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/19/Aachen_14-10-10_July_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/19/Aachen_14-10-10_July_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     </div>
     </div>
Line 50: Line 50:
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/August" style="color:black">
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/August" style="color:black">
-
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel">Go to August</div></div>
+
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to August</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4e/Aachen_14-10-10_August_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4e/Aachen_14-10-10_August_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     </div>
     </div>
Line 66: Line 66:
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
  <li style="width:130px;margin-left: 0px;margin-right: 0px;" >
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/October" style="color:black">
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/October" style="color:black">
-
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel">Go to October</div></div>
+
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to October</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/6/60/Aachen_14-10-10_October_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/6/60/Aachen_14-10-10_October_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
     </div>
     </div>

Revision as of 11:12, 14 October 2014

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


Contact Disclaimer

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