Team:TU Delft/23 July 2010 content

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=Surprise=
 +
We are very grateful for the help of our superior, Esengül in the lab. To thank her, we organized a surprise for her… In the afternoon a special delivery for Esengül arrived. Inside was a self-baked cake from a secret admirer. What a surprise this was! Half a day later, she found out that we were behind it and we all enjoyed eating the cake.
 +
 +
=Lab work=
 +
==Alkane Degradation==
==Alkane Degradation==
-
With a number of [https://2010.igem.org/Team:TU_Delft#/blog?blog=22_July_2010 yesterday's] colonies a digestion was done to check if they contain the desired insert. The table below describes the digestion procedure, as well as the gel lane description.
+
With a number of [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=22_July_2010 yesterday's] colonies a digestion was done to check if they contain the desired insert. The table below describes the [https://2010.igem.org/Team:TU_Delft/protocols/restriction_enzyme_digestion digestion] procedure, as well as the gel lane description. (Digestion was done in 10 μL end-volume)
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|'''#'''
|'''#'''
-
|'''Contents'''
+
|'''Sample'''
|'''Enzyme 1'''
|'''Enzyme 1'''
|'''Enzyme 2'''
|'''Enzyme 2'''
|'''Buffer'''
|'''Buffer'''
|'''BSA'''
|'''BSA'''
-
|'''Expected size(s) (bp)'''
 
|-
|-
|1
|1
-
|Smartladder (5μl)
 
-
|
 
-
|
 
-
|
 
-
|
 
-
|n/a
 
-
|-
 
-
|2
 
|007T (2)
|007T (2)
-
|0.5 μl EcoRI
+
|0.5 μL EcoRI
-
|0.5 μl PstI
+
|0.5 μL PstI
|3 (Biolabs)
|3 (Biolabs)
-
|no
+
|✗
-
|1341, 2455
+
|-
|-
-
|3
+
|2
|008T (4)
|008T (4)
-
|0.5 μl EcoRI
+
|0.5 μL EcoRI
-
|0.5 μl PstI
+
|0.5 μL PstI
|3 (Biolabs)
|3 (Biolabs)
-
|no
+
|✗
-
|276, 2455
+
|-
|-
-
|4
+
|3
|009T (6)
|009T (6)
-
|0.5 μl EcoRI
+
|0.5 μL EcoRI
-
|0.5 μl PstI
+
|0.5 μL PstI
|3 (Biolabs)
|3 (Biolabs)
-
|no
+
|✗
-
|285, 2455
+
|-
|-
-
|5
+
|4
|009T (7)
|009T (7)
-
|0.5 μl EcoRI
+
|0.5 μL EcoRI
-
|0.5 μl PstI
+
|0.5 μL PstI
|3 (Biolabs)
|3 (Biolabs)
-
|no
+
|✗
-
|285, 2455
+
|-
|-
-
|6
+
|5
|010T (9)
|010T (9)
-
|0.5 μl EcoRI
+
|0.5 μL EcoRI
-
|0.5 μl PstI
+
|0.5 μL PstI
|3 (Biolabs)
|3 (Biolabs)
-
|no
+
|✗
-
|1382, 2455
+
|-
|-
-
|7
+
|6
|017T (14)
|017T (14)
-
|0.5 μl EcoRI
+
|0.5 μL EcoRI
-
|0.5 μl PstI
+
|0.5 μL PstI
|3 (Biolabs)
|3 (Biolabs)
-
|no
+
|✗
-
|855, 2455
+
|-
|-
-
|8
+
|7
-
|Empty
+
-
|
+
-
|
+
-
|
+
-
|
+
-
|
+
-
|-
+
-
|9
+
|007T (2)
|007T (2)
-
|1 μl PstI
+
|1 μL PstI
-
|1 μl PvuII
+
|1 μL PvuII
|H (Boehringer)
|H (Boehringer)
-
|no
+
|✗
-
|2529,983,251
+
|-
|-
-
|10
+
|8
|008T (4)
|008T (4)
-
|1 μl AatII
+
|1 μL AatII
-
|0.5 μl KpnI
+
|0.5 μL KpnI
|A (Boehringer)
|A (Boehringer)
-
|1 μl
+
|✓
-
|271, 2427
+
|-
|-
-
|11
+
|9
|009T (6)
|009T (6)
-
|0.5 μl KpnI
+
|0.5 μL KpnI
-
|1 μl NruI
+
|1 μL NruI
|B (Boehringer)
|B (Boehringer)
-
|1 μl
+
|✓
-
|598, 2109
+
|-
|-
-
|12
+
|10
|009T (7)
|009T (7)
-
|0.5 μl KpnI
+
|0.5 μL KpnI
-
|1 μl NruI
+
|1 μL NruI
|B (Boehringer)
|B (Boehringer)
-
|1 μl
+
|✓
-
|598, 2109
+
|-
|-
-
|13
+
|11
|010T (9)
|010T (9)
-
|1 μl EarI
+
|1 μL EarI
-
|0.5 μl PstI
+
|0.5 μL PstI
|1 (Biolabs)
|1 (Biolabs)
-
|no
+
|✗
-
|1075, 2729
+
|-
|-
-
|14
+
|12
|017T (14)
|017T (14)
-
|0.5 μl AdhI
+
|0.5 μL AdhI
-
|0.5 μl SpeI
+
|0.5 μL SpeI
|4 (Biolabs)
|4 (Biolabs)
-
|no
+
|✗
-
|428, 2849
+
|}
 +
 
 +
[[Image:TUDelft_Digestion_Kira_23-07.png|550px|thumb|left|1% Agarose gel plasmid check using digestion reactions
 +
gel runned at 100V for 1 hour. Of all samples 5 μL + 1 μL loadingbuffer was loaded. 5 μL was loaded of marker]]
 +
 
 +
Lane description:
 +
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Expected Length (bp)'''
 +
|'''Status'''
|-
|-
-
|15
+
|M1
-
|EZ load (ladder)
+
|[https://2010.igem.org/Image:TU_Delft_SmartLadder.jpg SmartLadder]
 +
|n/a
 +
|n/a
 +
|-
 +
|1
 +
|007T (2) + EcoRI + PstI
 +
|1341, 2455
|
|
 +
|-
 +
|2
 +
|008T (4) + EcoRI + PstI
 +
|276, 2455
|
|
 +
|-
 +
|3
 +
|009T (6) + EcoRI + PstI
 +
|285, 2455
|
|
 +
|-
 +
|4
 +
|009T (7) + EcoRI + PstI
 +
|285, 2455
|
|
 +
|-
 +
|5
 +
|010T (9) + EcoRI + PstI
 +
|1382, 2455
 +
|
 +
|-
 +
|6
 +
|017T (14) + EcoRI + PstI
 +
|855, 2455
 +
|
 +
|-
 +
|7
 +
|Empty
 +
|
 +
|
 +
|-
 +
|8
 +
|007T (2) + PstI + PvuII
 +
|2529,983,251
 +
|
 +
|-
 +
|9
 +
|008T (4) + AatII + KpnI
 +
|271, 2427
 +
|
 +
|-
 +
|10
 +
|009T (6) + KpnI + NruI
 +
|598, 2109
 +
|
 +
|-
 +
|11
 +
|009T (7) + KpnI + NruI
 +
|598, 2109
 +
|
 +
|-
 +
|12
 +
|010T (9) + EarI + PstI
 +
|1075, 2729
 +
|
 +
|-
 +
|13
 +
|017T (14) + AdhI + SpeI
 +
|428, 2849
 +
|
 +
|-
 +
|M2
 +
|EZ load (ladder)
|n/a
|n/a
 +
|n/a
 +
|}
 +
 +
It seems (from the gel) that BioBrick 007T is the correct one, and the others don't contain the desired insert.
 +
 +
==Salt tolerance==
 +
 +
The paltes transformed on wednesday had colonies but they grew very slowly. Thus they were grown up for another night and were Sc PCR'd today.
 +
[[Image:Tudelft_ScPCR_bbc1-B0015.jpg|400px]]
 +
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''slot #'''
 +
|'''Contents'''
 +
|'''Result'''
|-
|-
 +
|1
 +
|NC (RFP from resealing plasmids)
 +
|
 +
|-
 +
|2
 +
|Ez Load
 +
|
 +
|-
 +
|3
 +
|SC Sample 1
 +
|Possibly positive
 +
|-
 +
|4
 +
|Sc Sample 2
 +
|Negative
 +
|-
 +
|5
 +
|Sc Sample 3
 +
|Negative
 +
|-
 +
|6
 +
|Sc Sample 4
 +
|Possibly positive
 +
|-
 +
|7
 +
|SC Sample 5
 +
|Possibly positive
 +
|-
 +
|8
 +
|Smartladder
|}
|}
-
[[Image:TUDelft_Digestion_Kira_23-07.png|550px|thumb|left|Digestion check]]
 
-
It seems (from the gel) that BioBrick 007T is the correct one, and the others don't contain the desired insert.
+
Here you can see that 3 samples are possibly positive, these will be sequenced and used for further steps in this process.

Latest revision as of 11:18, 10 August 2010

Contents

Surprise

We are very grateful for the help of our superior, Esengül in the lab. To thank her, we organized a surprise for her… In the afternoon a special delivery for Esengül arrived. Inside was a self-baked cake from a secret admirer. What a surprise this was! Half a day later, she found out that we were behind it and we all enjoyed eating the cake.

Lab work

Alkane Degradation

With a number of yesterday's colonies a digestion was done to check if they contain the desired insert. The table below describes the digestion procedure, as well as the gel lane description. (Digestion was done in 10 μL end-volume)

# Sample Enzyme 1 Enzyme 2 Buffer BSA
1 007T (2) 0.5 μL EcoRI 0.5 μL PstI 3 (Biolabs) ✗
2 008T (4) 0.5 μL EcoRI 0.5 μL PstI 3 (Biolabs) ✗
3 009T (6) 0.5 μL EcoRI 0.5 μL PstI 3 (Biolabs) ✗
4 009T (7) 0.5 μL EcoRI 0.5 μL PstI 3 (Biolabs) ✗
5 010T (9) 0.5 μL EcoRI 0.5 μL PstI 3 (Biolabs) ✗
6 017T (14) 0.5 μL EcoRI 0.5 μL PstI 3 (Biolabs) ✗
7 007T (2) 1 μL PstI 1 μL PvuII H (Boehringer) ✗
8 008T (4) 1 μL AatII 0.5 μL KpnI A (Boehringer) ✓
9 009T (6) 0.5 μL KpnI 1 μL NruI B (Boehringer) ✓
10 009T (7) 0.5 μL KpnI 1 μL NruI B (Boehringer) ✓
11 010T (9) 1 μL EarI 0.5 μL PstI 1 (Biolabs) ✗
12 017T (14) 0.5 μL AdhI 0.5 μL SpeI 4 (Biolabs) ✗
1% Agarose gel plasmid check using digestion reactions gel runned at 100V for 1 hour. Of all samples 5 μL + 1 μL loadingbuffer was loaded. 5 μL was loaded of marker

Lane description:

# Description Expected Length (bp) Status
M1 SmartLadder n/a n/a
1 007T (2) + EcoRI + PstI 1341, 2455
2 008T (4) + EcoRI + PstI 276, 2455
3 009T (6) + EcoRI + PstI 285, 2455
4 009T (7) + EcoRI + PstI 285, 2455
5 010T (9) + EcoRI + PstI 1382, 2455
6 017T (14) + EcoRI + PstI 855, 2455
7 Empty
8 007T (2) + PstI + PvuII 2529,983,251
9 008T (4) + AatII + KpnI 271, 2427
10 009T (6) + KpnI + NruI 598, 2109
11 009T (7) + KpnI + NruI 598, 2109
12 010T (9) + EarI + PstI 1075, 2729
13 017T (14) + AdhI + SpeI 428, 2849
M2 EZ load (ladder) n/a n/a

It seems (from the gel) that BioBrick 007T is the correct one, and the others don't contain the desired insert.

Salt tolerance

The paltes transformed on wednesday had colonies but they grew very slowly. Thus they were grown up for another night and were Sc PCR'd today. Tudelft ScPCR bbc1-B0015.jpg

slot # Contents Result
1 NC (RFP from resealing plasmids)
2 Ez Load
3 SC Sample 1 Possibly positive
4 Sc Sample 2 Negative
5 Sc Sample 3 Negative
6 Sc Sample 4 Possibly positive
7 SC Sample 5 Possibly positive
8 Smartladder


Here you can see that 3 samples are possibly positive, these will be sequenced and used for further steps in this process.

Retrieved from "http://2010.igem.org/Team:TU_Delft/23_July_2010_content"