TUDelft/21 October 2008

From 2008.igem.org

(Difference between revisions)
(New page: {{Template:TUDelftiGEM2008}} {{Template:TUDelftiGEM2008_menu_home}} {{Template:TUDelftiGEM2008_calendar}} =October 21st= ==New way for cell lysis: Beadbeater== ==Concentrating DNA for ...)
(→Growing Cells)
 
(8 intermediate revisions not shown)
Line 5: Line 5:
=October 21st=
=October 21st=
-
==New way for cell lysis: Beadbeater==
+
==New way for cell lysis: Bead Beater==
 +
A new protocol for cell lysis was tested: after 1ml of o/n LB culture (OD=~1) is resuspended in 1ml of PBS, 0.5g of small acid-washed glass beads and 20ul of 2uM lysozyme is added. The eppendorf tubes are shaked for 1h in the cold room on the Bead Beater. After this 600ul of the suspension is transferred to a fresh eppendorf tube. This is used to measure protein content and luciferase expression.
 +
 +
EDIT: Later this day, when protein content was measured, it was clear too much lysozyme was added. The concentration of the stock solution was too high. In figure 1 this can be seen. The maximum of the BCA measurement is 2 mg/ml, which we measure in every sample here. After we did some recalculation we estimated the concentration lysozyme was approximately 2 mg/ml. The experiment will be repeated.
 +
 +
 +
[[Image:TUDelft211008.jpg|550px|TUDelft071008.jpg]]
 +
Figure 1: BCA of samples treated with lysozyme and beadbeater.
==Concentrating DNA for sequencing==
==Concentrating DNA for sequencing==
 +
 +
DNA in samples that had a total concentration of less than 100ng/ul was precipitated. This was done by adding 1/10 volume NaAc and 2.5 volumes 96% EtOH. This was stored at -20oC for several hours (at least 1h). Samples were centrifuged 20' @ max speed (14,000 rpm) and pellets washed with 1.5 volumes 70% EtOH. Again, samples were spun off (10' @ max) and resuspended in the desired volume of water (alternatively, TE buffer could also be used).
 +
 +
==Growing Cells==
 +
 +
Just like yesterday, K115012, K115035 and K115036 were grown o/n @ 30ºC. These tubes will be used to test the Bead Beater protocol properly.
{{Template:TUDelftiGEM2008_sidebar}}
{{Template:TUDelftiGEM2008_sidebar}}

Latest revision as of 12:37, 29 October 2008

July
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31
August
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

September
MTWTFSS
[http://2008.igem.org/TUDelft/1_September_2008 1] [http://2008.igem.org/TUDelft/2_September_2008 2] [http://2008.igem.org/TUDelft/3_September_2008 3] [http://2008.igem.org/TUDelft/4_September_2008 4] [http://2008.igem.org/TUDelft/5_September_2008 5] [http://2008.igem.org/wiki/index.php?title=TUDelft/6_September_2008&action=edit 6] [http://2008.igem.org/wiki/index.php?title=TUDelft/7_September_2008&action=edit 7]
[http://2008.igem.org/TUDelft/8_September_2008 8] [http://2008.igem.org/TUDelft/9_September_2008 9] [http://2008.igem.org/TUDelft/10_September_2008 10] [http://2008.igem.org/TUDelft/11_September_2008 11] [http://2008.igem.org/TUDelft/12_September_2008 12] [http://2008.igem.org/wiki/index.php?title=TUDelft/13_September_2008&action=edit 13] [http://2008.igem.org/wiki/index.php?title=TUDelft/14_September_2008&action=edit 14]
[http://2008.igem.org/TUDelft/15_September_2008 15] [http://2008.igem.org/TUDelft/16_September_2008 16] [http://2008.igem.org/TUDelft/17_September_2008 17] [http://2008.igem.org/TUDelft/18_September_2008 18] [http://2008.igem.org/TUDelft/19_September_2008 19] [http://2008.igem.org/wiki/index.php?title=TUDelft/20_September_2008&action=edit 20] [http://2008.igem.org/wiki/index.php?title=TUDelft/21_September_2008&action=edit 21]
[http://2008.igem.org/TUDelft/22_September_2008 22] [http://2008.igem.org/TUDelft/23_September_2008 23] [http://2008.igem.org/TUDelft/24_September_2008 24] [http://2008.igem.org/TUDelft/25_September_2008 25] [http://2008.igem.org/wiki/index.php?title=TUDelft/26_September_2008&action=edit 26] [http://2008.igem.org/wiki/index.php?title=TUDelft/27_September_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=TUDelft/28_September_2008&action=edit 28]
[http://2008.igem.org/TUDelft/29_September_2008 29] [http://2008.igem.org/TUDelft/30_September_2008 30]
October
MTWTFSS
    [http://2008.igem.org/TUDelft/1_October_2008 1] [http://2008.igem.org/TUDelft/2_October_2008 2] [http://2008.igem.org/TUDelft/3_October_2008 3] [http://2008.igem.org/wiki/index.php?title=TUDelft/4_October_2008&action=edit 4] [http://2008.igem.org/wiki/index.php?title=TUDelft/5_October_2008&action=edit 5]
[http://2008.igem.org/TUDelft/6_October_2008 6] [http://2008.igem.org/TUDelft/7_October_2008 7] [http://2008.igem.org/TUDelft/8_October_2008 8] [http://2008.igem.org/TUDelft/9_October_2008 9] [http://2008.igem.org/TUDelft/10_October_2008 10] [http://2008.igem.org/wiki/index.php?title=TUDelft/11_October_2008&action=edit 11] [http://2008.igem.org/wiki/index.php?title=TUDelft/12_October_2008&action=edit 12]
[http://2008.igem.org/TUDelft/13_October_2008 13] [http://2008.igem.org/TUDelft/14_October_2008 14] [http://2008.igem.org/TUDelft/15_October_2008 15] [http://2008.igem.org/TUDelft/16_October_2008 16] [http://2008.igem.org/TUDelft/17_October_2008 17] [http://2008.igem.org/wiki/index.php?title=TUDelft/18_October_2008&action=edit 18] [http://2008.igem.org/wiki/index.php?title=TUDelft/19_October_2008&action=edit 19]
[http://2008.igem.org/TUDelft/20_October_2008 20] [http://2008.igem.org/TUDelft/21_October_2008 21] [http://2008.igem.org/TUDelft/22_October_2008 22] [http://2008.igem.org/TUDelft/23_October_2008 23] [http://2008.igem.org/TUDelft/24_October_2008 24] [http://2008.igem.org/wiki/index.php?title=TUDelft/25_October_2008&action=edit 25] [http://2008.igem.org/wiki/index.php?title=TUDelft/26_October_2008&action=edit 26]
[http://2008.igem.org/wiki/index.php?title=TUDelft/27_October_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=TUDelft/28_October_2008&action=edit 28] [http://2008.igem.org/wiki/index.php?title=TUDelft/29_October_2008&action=edit 29] [http://2008.igem.org/wiki/index.php?title=TUDelft/30_October_2008&action=edit 30] [http://2008.igem.org/wiki/index.php?title=TUDelft/31_October_2008&action=edit 31]

Contents

October 21st

New way for cell lysis: Bead Beater

A new protocol for cell lysis was tested: after 1ml of o/n LB culture (OD=~1) is resuspended in 1ml of PBS, 0.5g of small acid-washed glass beads and 20ul of 2uM lysozyme is added. The eppendorf tubes are shaked for 1h in the cold room on the Bead Beater. After this 600ul of the suspension is transferred to a fresh eppendorf tube. This is used to measure protein content and luciferase expression.

EDIT: Later this day, when protein content was measured, it was clear too much lysozyme was added. The concentration of the stock solution was too high. In figure 1 this can be seen. The maximum of the BCA measurement is 2 mg/ml, which we measure in every sample here. After we did some recalculation we estimated the concentration lysozyme was approximately 2 mg/ml. The experiment will be repeated.


TUDelft071008.jpg Figure 1: BCA of samples treated with lysozyme and beadbeater.

Concentrating DNA for sequencing

DNA in samples that had a total concentration of less than 100ng/ul was precipitated. This was done by adding 1/10 volume NaAc and 2.5 volumes 96% EtOH. This was stored at -20oC for several hours (at least 1h). Samples were centrifuged 20' @ max speed (14,000 rpm) and pellets washed with 1.5 volumes 70% EtOH. Again, samples were spun off (10' @ max) and resuspended in the desired volume of water (alternatively, TE buffer could also be used).

Growing Cells

Just like yesterday, K115012, K115035 and K115036 were grown o/n @ 30ºC. These tubes will be used to test the Bead Beater protocol properly.

Retrieved from "http://2008.igem.org/TUDelft/21_October_2008"