Team:Aachen/Notebook/Wetlab/April

From 2014.igem.org

(Difference between revisions)
(→April)
Line 20: Line 20:
== 12th ==
== 12th ==
-
* chemically competent NEB Top10, DH5α and BL21 were made
+
* Chemically competent 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
-
* efficiency of our competent cells was tested
+
* The efficiency of our competent cells was tested
-
** counted colonies on the agar plates 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
<center>
<center>
Line 40: Line 40:
* 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
Line 93: Line 93:
== 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
Line 108: Line 108:
| '''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>
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<center>
<center>
{| class="wikitable"
{| class="wikitable"
-
!  !!volume [µL]
+
!  !!Volume [µL]
|-
|-
| '''HF'''||10
| '''HF'''||10
Line 132: Line 132:
| '''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
Line 157: Line 157:
| '''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 were 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
Line 187: Line 187:
* '''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>

Revision as of 13:49, 13 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 were 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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