Team:Aachen/Notebook/Wetlab/April

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{| class="wikitable"
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|align="left"| [[Team:Aachen/Notebook/Wetlab/March|previous month]] ||align="center"| [[Team:Aachen/Notebook/Wetlab2|Wetlab main page]] || align="right"| [[Team:Aachen/Notebook/Wetlab/May|next month]]
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<!--  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/March" style="color:black">
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    <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>
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/April" style="color:black">
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    <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>
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    <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;">
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/May" style="color:black">
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/June" style="color:black">
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/July" style="color:black">
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    <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>
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  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/August" style="color:black">
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    <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>
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/September" style="color:black">
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    <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to September</div></div>
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/October" style="color:black">
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    <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>
 +
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= April =
= April =
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'''Organization'''
'''Organization'''
-
where to get:
+
Getting all kind of lab equipment
* key cards and keys for the lab
* key cards and keys for the lab
* cooled centifuge
* cooled centifuge
-
* space in -80&nbsp;°C
+
* space reservation in -80°C freezer
-
* bottles for 70&nbsp;% ethanol
+
* bottles for 70% ethanol
* ... (problems of a first year team ^^)
* ... (problems of a first year team ^^)
== 12th ==
== 12th ==
-
* chemically competent NEB Top10, DH5α and BL21 were made
+
* chemically competent ''E. coli'' NEB Top10, DH5α and BL21 were made
== 15th ==
== 15th ==
* 800&nbsp;ml LB for plates with 1.5% agar and kanamycin (50&nbsp;µg/L) were prepared
* 800&nbsp;ml LB for plates with 1.5% agar and kanamycin (50&nbsp;µg/L) were prepared
-
* efficiency of our competent cells was tested
+
* test of transformation efficiency of our competent cells
-
** counted colonies on the agar plates after transformation with 1&nbsp;µl DNA (147&nbsp;ng/µl)
+
** Colonies on the agar plates were counted after transformation with 1&nbsp;µl DNA (147&nbsp;ng/µl)
-
** cells had been incubated in SOC or LB medium
+
** Cells had been incubated in SOC or LB medium
<center>
<center>
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|  '''DH5α''' || SOC: 30 LB: 10 ||SOC: 7 LB: 1 || SOC: 1 LB: 2 || SOC: 2 LB: 1 ||
|  '''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
+
| '''NEB 10β''' || SOC: 170 LB: 135 ||SOC: 100 LB: 79 || SOC: 25 LB: 22 || SOC: 9 LB: 9 || SOC: 1 LB:0
|-
|-
|}
|}
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* BL21 cells were centrifuged and plated; only 128 colonies grew in total indicating a very bad efficiency (~482.11).
* 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:
+
Following formula was used to calculate the efficiency: ''Efficiency = (CFU /µg DNA) / Dilution''
-
efficiency = (CFU /µg DNA) / dilution
+
* for DH5α: medial efficiency in SOC: 1864.36, and in LB: 440.68
* 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
* for NEB:  medial efficiency in SOC: 6779.66, and in LB: 4698.30
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'''Pcq lab PCR'''  advanced primus 25, 96  
'''Pcq lab PCR'''  advanced primus 25, 96  
-
<center>
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<div style="float:left;margin-right:1em;">
{| class="wikitable"
{| class="wikitable"
!  !! Lenght [bp] !! % GC !! T<sub>m</sub> !! T<sub>A</sub>
!  !! Lenght [bp] !! % GC !! T<sub>m</sub> !! T<sub>A</sub>
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| '''REACh2_N''' || 320 || 59 || 82 || 54  
| '''REACh2_N''' || 320 || 59 || 82 || 54  
|-
|-
-
|}
+
|} </div>
-
</center>
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<div style="float:left;margin-right:1em;">
-
 
+
-
Program name IGEM.cyc
+
-
 
+
-
<center>
+
{| class="wikitable"
{| class="wikitable"
! Parameter !! Duration !! Temp [°C]
! Parameter !! Duration !! Temp [°C]
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|-
|-
| Store||forever||8
| Store||forever||8
-
|}
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|} </div>
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</center>
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# RFC25_F RFC25_R
# RFC25_F RFC25_R
Line 93: Line 169:
== 23th ==
== 23th ==
-
* 5&nbsp;µl of the PCR products were run on a 1,5% agarose gel
+
* 5&nbsp;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 contaminated with REACh1
+
* DNA bands were purified from gel with the High Pure Product Purification Kit. Full length products were contaminated with REACh1
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| '''dNTP'''||4||4
| '''dNTP'''||4||4
|-
|-
-
| '''template'''||2x 1||10
+
| '''Template'''||2x 1||10
|-
|-
| '''Phusion'''||0.5||0.5
| '''Phusion'''||0.5||0.5
|-
|-
-
| '''primer'''||-||2x 2.5
+
| '''Primer'''||-||2x 2.5
|-
|-
-
| '''water'''||33.5||21.5
+
| '''Water'''||33.5||21.5
|}
|}
</center>
</center>
-
'''Anneling''': 52&nbsp;°C
+
'''Anneling''': 52°C
'''Elongation''': 20 sec
'''Elongation''': 20 sec
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<center>
<center>
{| class="wikitable"
{| class="wikitable"
-
!  !!volume [µL]
+
!  !!Volume [µL]
|-
|-
| '''HF'''||10
| '''HF'''||10
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| '''dNTP'''||4
| '''dNTP'''||4
|-
|-
-
| '''template'''||1
+
| '''Template'''||1
|-
|-
| '''Phusion'''||0.5
| '''Phusion'''||0.5
|-
|-
-
| '''primer'''||2x 2.5
+
| '''Primer'''||2x 2.5
|-
|-
-
| '''water'''||29.5
+
| '''Water'''||29.5
|-
|-
-
| '''total'''||50
+
| '''Total'''||50
|}
|}
</center>
</center>
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<center>
<center>
{| class="wikitable"
{| class="wikitable"
-
!  !!volume
+
!  !!Volume
|-
|-
-
| '''destination vector'''||500&nbsp;ng
+
| '''Destination Vector'''||500&nbsp;ng
|-
|-
| '''EcoRI-HF'''||1&nbsp;µL
| '''EcoRI-HF'''||1&nbsp;µL
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| '''10x NEB Buffer 2.1'''||5&nbsp;µL
| '''10x NEB Buffer 2.1'''||5&nbsp;µL
|-
|-
-
| '''water'''||???&nbsp;µL
+
| '''Water'''||???&nbsp;µL
|-
|-
-
| '''total'''||???&nbsp;µL
+
| '''Total'''||???&nbsp;µL
|}
|}
</center>
</center>
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== 28th ==
== 28th ==
-
* measured the DNA concentration of SOE2 after purification
+
* The DNA concentration of the SOE2 products were measured after purification
* 1: 57&nbsp;ng/µL
* 1: 57&nbsp;ng/µL
* 2: 69.5&nbsp;ng/µL
* 2: 69.5&nbsp;ng/µL
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== 30th ==
== 30th ==
-
* PAGE of the SOE2 products were run on agarose gel for checking
+
* A PAGE of the SOE2 products was run on agarose gel for checking
*: &rarr; no positive results; only full length products were amplified
*: &rarr; no positive results; only full length products were amplified
-
* SOE PCRs was run again
+
* SOE PCRs were run again
*: &rarr; more template
*: &rarr; more template
*: &rarr; hot start
*: &rarr; hot start
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* '''SOE1'''
* '''SOE1'''
-
template A+B &rarr; 20&nbsp;µL &rarr; <u>13.8 + 6.2</u> <u>5.3 + 14.7</u>
+
Template A+B &rarr; 20&nbsp;µL &rarr; <u>13.8 + 6.2</u> <u>5.3 + 14.7</u>
<center>
<center>
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| '''dNTP'''||4
| '''dNTP'''||4
|-
|-
-
| '''template'''||20
+
| '''Template'''||20
|-
|-
| '''Phusion'''||0.5
| '''Phusion'''||0.5
|-
|-
-
| '''water'''||13.5
+
| '''Water'''||13.5
|}
|}
</center>
</center>
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* '''SOE2'''
* '''SOE2'''
-
 
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{| class="wikitable"
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<ul class="menusmall-grid">
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|align="left"| [[Team:Aachen/Notebook/Wetlab/March|previous month]] ||align="center"| [[Team:Aachen/Notebook/Wetlab2|Wetlab main page]] || align="right"| [[Team:Aachen/Notebook/Wetlab/May|next month]]
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<!--  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/March" style="color:black">
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    <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>
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    <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;">
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/April" style="color:black">
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    <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;">
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/May" style="color:black">
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    <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;">
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    <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" 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;">
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    <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" 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;">
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  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
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    <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" 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;">
 +
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 +
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 +
 
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  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/September" style="color:black">
 +
    <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to September</div></div>
 +
    <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/d/d4/Aachen_14-10-10_September_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
 +
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 +
  </li>
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 +
  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
 +
    <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" 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;">
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{{Team:Aachen/Footer}}
{{Team:Aachen/Footer}}

Latest revision as of 17:53, 17 October 2014

April

1st

Organization

Getting all kind of lab equipment

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

12th

  • chemically competent E. coli NEB Top10, DH5α and BL21 were made

15th

  • 800 ml LB for plates with 1.5% agar and kanamycin (50 µg/L) were prepared
  • test of transformation efficiency of our competent cells
    • 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 10β 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
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

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