Team:TU Delft/16 August 2010 content

From 2010.igem.org

(Difference between revisions)
(Digestion)
(Digestion)
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[[Image:TU_Delft_Digestion_16-08-10.png|400px|thumb|left|1% agarose of colony PCR. Gel runned at 100V for 1 hour. Of all samples 10 µL was loaded with 2 µL loadingbuffer. 2 µL was loaded of marker]]
[[Image:TU_Delft_Digestion_16-08-10.png|400px|thumb|left|1% agarose of colony PCR. Gel runned at 100V for 1 hour. Of all samples 10 µL was loaded with 2 µL loadingbuffer. 2 µL was loaded of marker]]
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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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|1
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|Smartladder
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|Varies
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|Yes
 +
|-
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|2
 +
|011A cut
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|<b>1523</b>, 1199, 857
 +
|Yes
 +
|-
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|3
 +
|013K cut
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|1825, <b>1592</b>, 1342, 43
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|Yes
 +
|-
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|4
 +
|017A cut
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|<b>1410</b>, 1199, 857
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|Yes
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|-
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|5
 +
|018A cut
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|1185, 872, <b>793</b>, 43
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|Yes
 +
|-
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|6
 +
|402A cut
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|1199, 857, <b>543</b>
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|Yes
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|-
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|7
 +
|403A cut
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|1185, 872, <b>397</b>, 43
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|Yes
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|-
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|8
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|pCaiF in pANY cut
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|1277, 1143, <b>74</b>
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|Yes
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|-
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|9
 +
|B0032 in pSB1A2 cut
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|1185, 872, <b>35</b>
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|Yes
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|-
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|10
 +
|B0032 in pSB1A2 cut
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|1185, 872, <b>35</b>
 +
|Yes
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|11
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|pSB1C3 cut
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|<b>2035</b>, 1081
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|Yes (some left uncut at 3116 bp)
 +
|}

Revision as of 19:01, 16 August 2010

Alkane degradation

Unfortunately last week's attempts hadn't yielded any BioBricks making use of the 2-way ligation method. We thus decided to take another stab at the 3-way ligation procedure, according to the BioBrick Assembly Manual of Ginkgo Bioworks. The BioBrick numbers we tried to construct were: 014C, 020C, 405C, 327C and 304C.

Digestion

# Sample Enzyme 1 Enzyme 2 Enzyme 3 Buffer BSA Needed fragment
1 011A EcoRI SpeI AseI NEBuffer 2 ‘E - J23100-J61100-alkB2-J61100-rubA3 - S’
2 013K XbaI PstI HindIII NEBuffer 2 ‘X - J61100-rubA4-J61100-rubR-B0015 - P’
3 017A EcoRI SpeI AseI NEBuffer 2 ‘E - J23100-J61100-ladA - S’
4 018A XbaI PstI AseI NEBuffer 2 ‘X - J61101-ADH - P’
5 402A EcoRI SpeI AseI NEBuffer 2 ‘E - J23100-J61101-PhPFDα - S’
6 403A XbaI PstI AseI NEBuffer 2 ‘X - J61101-PhPFDβ - P’
7 pCaiF EcoRI SpeI AseI NEBuffer 2 ‘E - pCaiF - S’
8 B0032 XbaI PstI AseI NEBuffer 2 ‘X - B0032 - P’
9 B0032 XbaI PstI AseI NEBuffer 2 ‘X - B0032 - P’
10 pAlkS EcoRI SpeI AseI NEBuffer 2 ‘E - pAlkS - P’
11 pSB1C3 EcoRI PstI None NEBuffer 2 ‘E - pSB1C3 - P’

The digestions were executed for 15 minutes at 37 degrees, and checked on 1% agarose gel:

1% agarose of colony PCR. Gel runned at 100V for 1 hour. Of all samples 10 µL was loaded with 2 µL loadingbuffer. 2 µL was loaded of marker

Lane description

# Description Expected size (bp) OK?
1 Smartladder Varies Yes
2 011A cut 1523, 1199, 857 Yes
3 013K cut 1825, 1592, 1342, 43 Yes
4 017A cut 1410, 1199, 857 Yes
5 018A cut 1185, 872, 793, 43 Yes
6 402A cut 1199, 857, 543 Yes
7 403A cut 1185, 872, 397, 43 Yes
8 pCaiF in pANY cut 1277, 1143, 74 Yes
9 B0032 in pSB1A2 cut 1185, 872, 35 Yes
10 B0032 in pSB1A2 cut 1185, 872, 35 Yes 11 pSB1C3 cut 2035, 1081 Yes (some left uncut at 3116 bp)