Team:TU Delft/6 July 2010 content

From 2010.igem.org

(Difference between revisions)
(Lab work)
(Lab work)
Line 158: Line 158:
Subsequent overnight [http://2010.igem.org/Team:TU_Delft/protocols/ligation ligation] yielded:
Subsequent overnight [http://2010.igem.org/Team:TU_Delft/protocols/ligation ligation] yielded:
-
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
+
| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|'''#'''
|'''#'''
-
|'''Ligation reaction'''
+
|'''BioBrick'''
 +
|'''Recipient plasmid'''
 +
|'''Fragment 2'''
 +
|'''Final volume'''
|-
|-
|1
|1
-
|3 μL I10341 + 4 μL J1100 + 1.5 μL pSB1C3
+
|K398500A
 +
|130 μg ‘S – J61100 - pSB1A2 – P’
 +
|154 μg ‘X-I13401-P’
 +
|26 μL
|-
|-
|2
|2
-
|3 μL I10341 + 4 μL J1101 + 1.5 μL pSB1C3
+
|K398501A
 +
|206 μg ‘S – J61101 - pSB1A2 – P’
 +
|247 μg ‘X-I13401-P’
 +
|25 μL
|-
|-
|3
|3
-
|3 μL I10341 + 4 μL J1107 + 1.5 μL pSB1C3
+
|K398502A
 +
|196 μg ‘S – J61107 - pSB1A2 – P’
 +
|247 μg ‘X-I13401-P’
 +
|25 μL
|-
|-
|4
|4
-
|3 μL I10341 + 4 μL J1117 + 1.5 μL pSB1C3
+
|K398503A
 +
|201 μg ‘S – J61117 - pSB1A2 – P’
 +
|247 μg ‘X-I13401-P’
 +
|25 μL
|-
|-
|5
|5
-
|3 μL I10341 + 4 μL J1127 + 1.5 μL pSB1C3
+
|K398504A
 +
|202 μg ‘S – J61127 - pSB1A2 – P’
 +
|247 μg ‘X-I13401-P’
 +
|25.5 μL
|-
|-
|6
|6
-
|4 μL J1100 + 1.5 μL pSB3C5 (negative control)
+
|Ligase Control (J61117 - pSB1A2)
 +
|123 μg ‘S – J61117 - pSB1A2 – P’
 +
|None
 +
|15 μL
 +
|-
|}
|}
 +
 +
To all samples one-tenth of the total volume ligase buffer was added.
<h4>Emulsifier</h4>
<h4>Emulsifier</h4>

Revision as of 13:28, 20 July 2010

Contents

Lab work

Ordered DNA stocks

The DNA from Mr Gene has finally arrived, now we're ready to build some biobricks!

We were so excited; we immediately dissolved the DNA in water and performed a transformation with 100 ng of the DNA and plated on the LB agar containing the appropriate antibiotic.

  • AlkB2 (Ampicillin)
  • rubA3 (Ampicillin)
  • rubA4 (Ampicillin)
  • rubB (Kanamycin)
  • ladA (Ampicillin)
  • ADH (Ampicillin)
  • ALDH (Kanamycin)
  • bbc1 (Ampicillin)
  • AlnA (Ampicillin)
  • OprG (Ampicillin)
  • PalkS1-2 (Ampicillin)
  • PalkB (Ampicillin)
  • P(CaiF) (Ampenicillin)
  • AlkS (Kanamycin)
  • PhPFD-α (Ampicillin)
  • PhPFD-β (Ampicillin)

Characterization of Anderson RBS sequences

Yesterday's purified PCR product of I13401 and the Anderson RBS Biobricks (J61100, J61101, J61107, J61117 and J61127) were digested:

# Digestion reaction Used Buffer Needed fragment
C1 1.15 μg J61100 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61100 - pSB1A2 – P’
C2 1.68 μg J61101 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61101 - pSB1A2 – P’
C3 1.30 μg J61107 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61107 - pSB1A2 – P’
C4 0.95 μg J61117 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61117 - pSB1A2 – P’
C5 0.51 μg J61127 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J61127 - pSB1A2 – P’
D1 2.59 μg I13401 + XbaI + PstI Buffer 2 (BioLabs) ‘X – I13401 – P’
D2 2.65 μg I13401 + XbaI + PstI Buffer 2 (BioLabs) ‘X – I13401 – P’

The digestion products were purified using Roche's PCR Purifiation Kit and loaded onto a 1% agarose gel for comparison with the non-digested BioBricks:

Digestion check

Lane description:

# Description Expected Length (bp) Remarks
1 SmartLadder marker (5 μL) n/a
2 Undigested J61100 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular Plasmid
3 Undigested J61101 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular Plasmid
4 ‘S – J61100 - pSB1A2 – P’ 2116 Visible
5 ‘S – J61101 - pSB1A2 – P’ 2116 Visible
6 Undigested J61107 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular plasmid
7 ‘S – J61107 - pSB1A2 – P’ 2116 Visible
8 Undigested J61117 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular plasmid
9 ‘S – J61117 - pSB1A2 – P’ 2116 Visible
10 Undigested J61127 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular plasmid
11 ‘S – J61127 - pSB1A2 – P’ 2116 Visible
12 Undigested I13401 (1 μL + 4 μL MQ + 1 μL Loading Buffer) Circular plasmid
13 ‘X – I13401 – P’ 883 Visible
14 empty empty empty
15 BioRad EZ Load n/a Visible

Subsequent overnight ligation yielded:

| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" |# |BioBrick |Recipient plasmid |Fragment 2 |Final volume |- |1 |K398500A |130 μg ‘S – J61100 - pSB1A2 – P’ |154 μg ‘X-I13401-P’ |26 μL |- |2 |K398501A |206 μg ‘S – J61101 - pSB1A2 – P’ |247 μg ‘X-I13401-P’ |25 μL |- |3 |K398502A |196 μg ‘S – J61107 - pSB1A2 – P’ |247 μg ‘X-I13401-P’ |25 μL |- |4 |K398503A |201 μg ‘S – J61117 - pSB1A2 – P’ |247 μg ‘X-I13401-P’ |25 μL |- |5 |K398504A |202 μg ‘S – J61127 - pSB1A2 – P’ |247 μg ‘X-I13401-P’ |25.5 μL |- |6 |Ligase Control (J61117 - pSB1A2) |123 μg ‘S – J61117 - pSB1A2 – P’ |None |15 μL |- |}

To all samples one-tenth of the total volume ligase buffer was added.

Emulsifier

Pieter is testing different condition for the emulsifying Assay. To determine the emulsifying activity we set up a calibration curve with SDS. He prepared the following samples:

# Hexane (mL) Tris Buffer pH 8 (mL) 10% SDS (mL)
B 1 1.1 0
100 1 1 0.1
50 1 1.05 0.05
25 1 1.075 0.025
10 1 1.09 0.01
5 1 1.095 0.005

Kampioenen!!!!

Today the Dutch team will play the semi-final of the World Cup! Hear us cheer! Stop sound

Pieter by the BBQ
We are ready for the soccer game
the Netherlands won the semi-final!