Team:TUDelft/13 August 2009

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='''13 August 2009'''=
='''13 August 2009'''=
 +
===Sriram===
 +
Today i understood that running the gel with ligated products is unnecessary which is clear in the below gel. I must not do this mistake again. I plated the transformed colonies on the plate.
 +
===Calin===
 +
 +
Knockout day. [http://openwetware.org/wiki/NanoBio:_Protocol_for_gene_knockout NanoBio: Protocol for gene knockout]
 +
 +
Made 1M l-arabinose stock. 150mg into 0.85 mL ddH20.
 +
 +
Digested CE with EcoRI (10uL DNA).
 +
 +
Yesterdays gel:
 +
 +
[[Image:Gel120809-delay-conj.png|thumb|center|550px]]
 +
 +
 +
{| border="1" align="center"
 +
| Well || Part || Expected Plasmid Size || Status
 +
|- align="center"
 +
| 1 ||  ||  ||
 +
|- align="center"
 +
| 2 ||  || ||
 +
|- align="center"
 +
| 3 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 4 || E0240 || 876 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 5 || B0031 || 14 || ?
 +
|- align="center"
 +
| 6 || J04630 || 857 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 7 || R0010 || 200 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 8 || R0040 || 54 || too small
 +
|- align="center"
 +
| 9 || pSB1C3 || 2072  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 10 || pSB1AK3 || 3426 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 11 || Assembly 1 || ||
 +
|- align="center"
 +
| 12 || Assembly 3 || ||
 +
|- align="center"
 +
| 13 || Assembly 5 || ||
 +
|- align="center"
 +
| 14 || Assembly 6 || ||
 +
|- align="center"
 +
| 15 || Assembly 7 || ||
 +
|- align="center"
 +
| 16 || Assembly 8 || ||
 +
|- align="center"
 +
| 17 || trbK X+P || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 18 || GFP-gen E+S || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 19 || pTet+RBS E+S || || ?
 +
|- align="center"
 +
| 20 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  || <font color=limegreen>&#10004;</font>
 +
|}
 +
 +
Made 12 plates. 4 High KAN. 4 Low KAN. 4 TRI.
 +
 +
Placed 50uL of overnight R751 + pKD46 culture into 4 x 5 mL tubes.
 +
 +
In 30C incubator at 11:15
 +
 +
11:45 0.034 OD
 +
 +
12:30 0.065 OD
 +
 +
13:17 0.126 OD
 +
 +
Induced +L-arabinose tubes with L-arabinose (40uL 1M stock added to 4mL). Control tube has no L-arabinose added.
 +
 +
In 30C incubator at 13:25
 +
 +
14:40 0.497 OD
 +
 +
on Ice at 14:57
 +
 +
centrifuge 1 @ 15:13 - 10 min 4000 rcf 3C
 +
 +
centrifuge 2 @ 15:44 - 10 min 4000 rcf 3C
 +
 +
centrifuge 3 @ 16:10 - 10 min 4000 rcf 3C
 +
 +
12 plates made, of which 8 are various controls
 +
 +
{| border="1" align="center"
 +
| Plate ID || Knockout || Variables || AB
 +
|- align="center"
 +
| 1H || oriTR_KO || +L-arabinose +PCR  || 1xKAN
 +
|- align="center"
 +
| 1L || oriTR_KO || +L-arabinose +PCR || 0.5xKAN
 +
|- align="center"
 +
| 2 || oriTR_KO || +L-arabinose -PCR || TRI
 +
|- align="center"
 +
| 3H || oriTR_KO || -L-arabinose +PCR || 1xKAN
 +
|- align="center"
 +
| 3L || oriTR_KO || -L-arabinose +PCR || 0.5xKAN
 +
|- align="center"
 +
| 4 || oriTR_KO || -L-arabinose -PCR || TRI
 +
|- align="center"
 +
| 5H || trbK_KO || +L-arabinose +PCR || 1xKAN
 +
|- align="center"
 +
| 5L || trbK_KO || +L-arabinose +PCR || 0.5xKAN
 +
|- align="center"
 +
| 6 || trbK_KO || +L-arabinose -PCR  || TRI
 +
|- align="center"
 +
| 7H || trbK_KO || -L-arabinose +PCR || 1xKAN
 +
|- align="center"
 +
| 7L || trbK_KO || -L-arabinose +PCR || 0.5xKAN
 +
|- align="center"
 +
| 8 || trbK_KO || -L-arabinose -PCR || TRI
 +
|}
 +
 +
Tim Vos helped with electroporation step. Electroporator was set to 600 ohm.
 +
 +
{| border="1" align="center"
 +
| Incubator Time || Knockout || Variables || Time Constant
 +
|- align="center"
 +
| 18:42 || oriTR_KO || +L-arabinose +PCR  || 12.0
 +
|- align="center"
 +
| 18:45 || oriTR_KO || +L-arabinose -PCR || 13.3
 +
|- align="center"
 +
| 18:48 || oriTR_KO || -L-arabinose +PCR || 10.4
 +
|- align="center"
 +
| 18:50 || oriTR_KO || -L-arabinose -PCR || 13.4
 +
|- align="center"
 +
| 18:54 || trbK_KO || +L-arabinose +PCR || 12.0
 +
|- align="center"
 +
| 18:56 || trbK_KO || +L-arabinose -PCR  || 12.9
 +
|- align="center"
 +
| 17:02 || trbK_KO || -L-arabinose +PCR || 13.0
 +
|- align="center"
 +
| 17:05 || trbK_KO || -L-arabinose -PCR || 12.6
 +
|}
 +
 +
After one hour 200 uL was plated. oriTR_KO +L-ara +PCR and trbK_KO +L-ara +PCR placed in fridge at 18:30.
 +
 +
Made 5mL tube cultures for assemblies CB and CC.
 +
 +
Todays 8 well gel to check the linear fragments for the knockout:
 +
 +
[[Image:Gel130809TimandCalin.png|thumb|center|250px]]
 +
 +
{| border="1" align="center"
 +
| Well || Part || Expected Plasmid Size || Status
 +
|- align="center"
 +
| 2 || CE || 2157  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 3 || oriTR_KO_PCR || 1596 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 4 || trbK_KO_PCR || 1596 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 5 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 6 || ? || || ?
 +
|}
 +
 +
===Daniel===
 +
 +
Yesterday´s gel confirm the sizes of all the biobricks needed (see the wells 4-10 in the first gel of this site), I did a second round of digestions though, in order to have enough DNA.
 +
After a new gel and check sizes, I did the assemblies 1A and 2A (see locks and keys section). Also, I transformed by heat shock and electroporation these assemblies and did plates and culture tubes with respective antibiotic.
 +
{{Template:TUDelftiGEM2009_end}}
{{Template:TUDelftiGEM2009_end}}

Latest revision as of 09:33, 20 October 2009

Lab Notebook

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
  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
October
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
 

13 August 2009

Sriram

Today i understood that running the gel with ligated products is unnecessary which is clear in the below gel. I must not do this mistake again. I plated the transformed colonies on the plate.

Calin

Knockout day. [http://openwetware.org/wiki/NanoBio:_Protocol_for_gene_knockout NanoBio: Protocol for gene knockout]

Made 1M l-arabinose stock. 150mg into 0.85 mL ddH20.

Digested CE with EcoRI (10uL DNA).

Yesterdays gel:

Gel120809-delay-conj.png


Well Part Expected Plasmid Size Status
1
2
3 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔
4 E0240 876 ✔
5 B0031 14  ?
6 J04630 857 ✔
7 R0010 200 ✔
8 R0040 54 too small
9 pSB1C3 2072 ✔
10 pSB1AK3 3426 ✔
11 Assembly 1
12 Assembly 3
13 Assembly 5
14 Assembly 6
15 Assembly 7
16 Assembly 8
17 trbK X+P ✔
18 GFP-gen E+S ✔
19 pTet+RBS E+S  ?
20 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔

Made 12 plates. 4 High KAN. 4 Low KAN. 4 TRI.

Placed 50uL of overnight R751 + pKD46 culture into 4 x 5 mL tubes.

In 30C incubator at 11:15

11:45 0.034 OD

12:30 0.065 OD

13:17 0.126 OD

Induced +L-arabinose tubes with L-arabinose (40uL 1M stock added to 4mL). Control tube has no L-arabinose added.

In 30C incubator at 13:25

14:40 0.497 OD

on Ice at 14:57

centrifuge 1 @ 15:13 - 10 min 4000 rcf 3C

centrifuge 2 @ 15:44 - 10 min 4000 rcf 3C

centrifuge 3 @ 16:10 - 10 min 4000 rcf 3C

12 plates made, of which 8 are various controls

Plate ID Knockout Variables AB
1H oriTR_KO +L-arabinose +PCR 1xKAN
1L oriTR_KO +L-arabinose +PCR 0.5xKAN
2 oriTR_KO +L-arabinose -PCR TRI
3H oriTR_KO -L-arabinose +PCR 1xKAN
3L oriTR_KO -L-arabinose +PCR 0.5xKAN
4 oriTR_KO -L-arabinose -PCR TRI
5H trbK_KO +L-arabinose +PCR 1xKAN
5L trbK_KO +L-arabinose +PCR 0.5xKAN
6 trbK_KO +L-arabinose -PCR TRI
7H trbK_KO -L-arabinose +PCR 1xKAN
7L trbK_KO -L-arabinose +PCR 0.5xKAN
8 trbK_KO -L-arabinose -PCR TRI

Tim Vos helped with electroporation step. Electroporator was set to 600 ohm.

Incubator Time Knockout Variables Time Constant
18:42 oriTR_KO +L-arabinose +PCR 12.0
18:45 oriTR_KO +L-arabinose -PCR 13.3
18:48 oriTR_KO -L-arabinose +PCR 10.4
18:50 oriTR_KO -L-arabinose -PCR 13.4
18:54 trbK_KO +L-arabinose +PCR 12.0
18:56 trbK_KO +L-arabinose -PCR 12.9
17:02 trbK_KO -L-arabinose +PCR 13.0
17:05 trbK_KO -L-arabinose -PCR 12.6

After one hour 200 uL was plated. oriTR_KO +L-ara +PCR and trbK_KO +L-ara +PCR placed in fridge at 18:30.

Made 5mL tube cultures for assemblies CB and CC.

Todays 8 well gel to check the linear fragments for the knockout:

Gel130809TimandCalin.png
Well Part Expected Plasmid Size Status
2 CE 2157 ✔
3 oriTR_KO_PCR 1596 ✔
4 trbK_KO_PCR 1596 ✔
5 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔
6  ?  ?

Daniel

Yesterday´s gel confirm the sizes of all the biobricks needed (see the wells 4-10 in the first gel of this site), I did a second round of digestions though, in order to have enough DNA. After a new gel and check sizes, I did the assemblies 1A and 2A (see locks and keys section). Also, I transformed by heat shock and electroporation these assemblies and did plates and culture tubes with respective antibiotic.

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