Team:TU Delft/10 August 2010 content

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(Difference between revisions)
(→Lab work)
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A new attempt to construct parts 304 (PalkS12 + B0032), 327(P(caif) + B0032) and 405 (402 + 403).
A new attempt to construct parts 304 (PalkS12 + B0032), 327(P(caif) + B0032) and 405 (402 + 403).
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===Digestion, Ligation, Transformation===
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1 microgram of each plasmid was digested:
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|'''#'''
|'''#'''
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|✓
|✓
|‘E - J61101 - PhPFDbeta - S’
|‘E - J61101 - PhPFDbeta - S’
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|}
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The digestion was checked on a gel:
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[[Image:TU_Delft_2010-08-10_digestion_p7.png]]
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Lane description
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|'''#'''
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|'''Description'''
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|'''Expected size (bp)'''
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|'''OK?'''
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|-
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|L
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|Smartladder (5μl)
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|n/a
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|n/a
 +
|-
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|1
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|PalkS cut
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|106, 861, 1188
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|no
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|-
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|2
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|P(caif) cut
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|74, 861, 118
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|no
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|-
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|4
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|402 cut
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|543, 548, 1508
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|no
 +
|-
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|5
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|403 cut
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|2439, 58
 +
|vague
|}
|}

Revision as of 12:23, 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?
L Smartladder (5μl) n/a n/a
1 PalkS cut 106, 861, 1188 no
2 P(caif) cut 74, 861, 118 no
4 402 cut 543, 548, 1508 no
5 403 cut 2439, 58 vague

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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