Team:Aachen/Notebook/Wetlab/April

From 2014.igem.org

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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 °C
+
* space reservation in -80°C freezer
-
* bottles for 70 % 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 ml LB for plates with 1.5% agar and kanamycin (50 µg/L) were prepared
* 800 ml LB for plates with 1.5% agar and kanamycin (50 µg/L) were prepared
-
* The efficiency of our competent cells was tested
+
* test of transformation efficiency of our competent cells
** Colonies on the agar plates were counted after transformation with 1 µl DNA (147 ng/µl)
** 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
** Cells had been incubated in SOC or LB medium
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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  
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<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  
|-
|-
-
|}
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|} </div>
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</center>
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<div style="float:left;margin-right:1em;">
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-
Program name IGEM.cyc
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-
 
+
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<center>
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{| 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>
-
</center>
+
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
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 +
 
 +
 
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 +
 
 +
 
# RFC25_F RFC25_R
# RFC25_F RFC25_R
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</center>
</center>
-
'''Anneling''': 52&nbsp;°C
+
'''Anneling''': 52°C
'''Elongation''': 20 sec
'''Elongation''': 20 sec

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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