Team:TU Delft/10 August 2010 content

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The digestion was checked on a gel:
The digestion was checked on a gel:
-
[[Image:TU_Delft_2010-08-10_digestion_p7.png]]
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[[Image:TU_Delft_2010-08-10_digestion_p7.png|200px]]
Lane description
Lane description
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|'''OK?'''
|'''OK?'''
|-
|-
-
|L
+
|1
|Smartladder (5μl)
|Smartladder (5μl)
|n/a
|n/a
|n/a
|n/a
|-
|-
-
|1
+
|2
|PalkS cut
|PalkS cut
|106, 861, 1188
|106, 861, 1188
|no
|no
|-
|-
-
|2
+
|3
|P(caif) cut
|P(caif) cut
|74, 861, 118
|74, 861, 118
|no
|no
|-
|-
-
|4
+
|5
|402 cut
|402 cut
|543, 548, 1508
|543, 548, 1508
|no
|no
|-
|-
-
|5
+
|6
|403 cut
|403 cut
|2439, 58
|2439, 58
|vague
|vague
 +
|}
 +
 +
So most digestions were not what we expected. The ligations were already started anyway.
 +
 +
Following the digestions, the fragments were [[Team:TU_Delft/protocols/ligation|ligated]] for 4 hours.
 +
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''BioBrick'''
 +
|'''Fragment 1'''
 +
|'''Recipient vector'''
 +
|-
 +
|1
 +
|304A
 +
|12.5 μL ‘E - PalkS12 - S’
 +
|10 μL ‘X - B0032 - E’
 +
|-
 +
|2
 +
|327A
 +
|10 μL ‘E - P(caif) – S’
 +
|10 μL ‘X - B0032 - E’
 +
|-
 +
|3
 +
|405A
 +
|15 μL ‘E - J61101 - PhPFDalpha – S’
 +
|3 μL ‘X - J61101 - PhPFDbeta - E’
|}
|}

Latest revision as of 12:31, 16 August 2010

Contents

Lab work

Tolerance and Sensing

A new attempt to construct parts 304 (PalkS12 + B0032), 327(P(caif) + B0032) and 405 (402 + 403).

Digestion, Ligation, Transformation

1 microgram of each plasmid was digested:

# Sample Enzyme 1 Enzyme 2 Enzyme 3 Buffer BSA Needed fragment
1 PalkS12A EcoRI SpeI ScaI 2 (Biolabs) ✓ ‘E - PalkS12 - S’
2 P(caif)A EcoRI SpeI ScaI 2 (Biolabs) ✓ ‘E - P(caif) - S’
3 B0032 EcoRI XbaI 2 (Biolabs) ✓ ‘X - B0032 - E’
4 402: J61101 - PhPFDalpha EcoRI SpeI ScaI 2 (Biolabs) ✓ ‘E - J61101 - PhPFDalpha - S’
5 403: J61101 - PhPFDbeta EcoRI XbaI 2 (Biolabs) ✓ ‘E - J61101 - PhPFDbeta - S’

The digestion was checked on a gel:

TU Delft 2010-08-10 digestion p7.png

Lane description

# Description Expected size (bp) OK?
1 Smartladder (5μl) n/a n/a
2 PalkS cut 106, 861, 1188 no
3 P(caif) cut 74, 861, 118 no
5 402 cut 543, 548, 1508 no
6 403 cut 2439, 58 vague

So most digestions were not what we expected. The ligations were already started anyway.

Following the digestions, the fragments were ligated for 4 hours.

# BioBrick Fragment 1 Recipient vector
1 304A 12.5 μL ‘E - PalkS12 - S’ 10 μL ‘X - B0032 - E’
2 327A 10 μL ‘E - P(caif) – S’ 10 μL ‘X - B0032 - E’
3 405A 15 μL ‘E - J61101 - PhPFDalpha – S’ 3 μL ‘X - J61101 - PhPFDbeta - E’

Alkane degradation

All of yesterday's plates contained colonies. To check whether they contain the correct insert we will do a colony PCR. TUDelft 20100810 PCR.png

Lane description

# Description Primers Expected length (bp) ✓ ✗
L SmartLadder n/a n/a n/a n/a
1-5 013A G00100 + G00101 1929 none all
6-10 013K G00100 + G00101 1929 9 6,7,8,10
11-15 020A G00100 + G00101 2482 none all
L SmartLadder n/a n/a n/a n/a

On the gel it looks like lane 9, containing 013K, has the correct length. We will do a plasmid isolation tomorrow. Unfortunately none of the colonies of 020A seemed to be correct. We will check some more colonies with a colony PCR overnight.

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