Team:Cambridge/LabBook/Week11

From 2010.igem.org

(Difference between revisions)
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<div align="center">Week 11: Monday 20th September - Sunday 26th September</div>
<div align="center">Week 11: Monday 20th September - Sunday 26th September</div>
==Monday==
==Monday==
 +
 +
==Tuesday==
===107. Expt: Biobrick assembly of pBAD+Luc (PP + LC) and (Emily)===
===107. Expt: Biobrick assembly of pBAD+Luc (PP + LC) and (Emily)===
*Plasmids already extracted
*Plasmids already extracted
Line 56: Line 58:
*Gel extraction - results in -20°C freezer
*Gel extraction - results in -20°C freezer
 +
==Wednesday==
===108. Expt: Send off PP + pSB1C3, EPIC pBAD for sequencing (Emily and Peter)===
===108. Expt: Send off PP + pSB1C3, EPIC pBAD for sequencing (Emily and Peter)===
*Miniprep EPIC pBAD and nanodrop
*Miniprep EPIC pBAD and nanodrop
*Prepare correct concentrations (100ng/µl for plasmid, 3.2pmol/µl for primers) to be sent off
*Prepare correct concentrations (100ng/µl for plasmid, 3.2pmol/µl for primers) to be sent off
 +
===109. Expt: Biobrick assembly of pBAD + PP Luc (1), PP Luc (2), LC Luc (1), PP+pSB1C3===
 +
*Peter 'miniprepped' PP+pSB1C3 to extract plasmid
 +
*pBAD was amplified after trying to grow overnight culture failed. The following protocol was used:
 +
Add to a PCR tube:
 +
{|class="wikitable"
 +
|-
 +
|10µl
 +
|2x Phusion Master Mix
 +
|-
 +
|1µl
 +
|VF2 Primer
 +
|-
 +
|1µl
 +
|VR Primer
 +
|-
 +
|8µl
 +
|HPLC H20
 +
|-
 +
|20µl
 +
|
 +
|}
 +
 +
*Use stab to extract colony from pBAD plate and swirl in tube. Run following program on PCR machine saved as 'pBAD Amplification':
 +
**Heated lid at 110°C
 +
**Denaturation for 1m30s at 98°C
 +
**Cycle 35 times
 +
***Denaturation for 30s at 98°C
 +
***Elongation for 2m at 72°C
 +
**Elongation for 7m30s at 72°C
 +
 +
*PCR Purification of pBAD from PCR reaction was performed using Qiagen kit
 +
*Restriction: repeating restriction of LC Luc (1) because tube from yesterday was dropped. Use protocol on p88 with following quantities (in µl):
 +
{|class="wikitable"
 +
|-
 +
|
 +
|pBAD
 +
|LC Luc (1)
 +
|-
 +
|HPLC H20
 +
|0
 +
|14
 +
|-
 +
|10x FD Buffer
 +
|2
 +
|2
 +
|-
 +
|Plasmid DNA
 +
|16 (PCR product)*
 +
|2
 +
|-
 +
|FD EcoRI
 +
|1
 +
|1
 +
|-
 +
|FD XbaI
 +
|0
 +
|1
 +
|-
 +
|FD SpeI
 +
|1
 +
|0
 +
|}
 +
 +
'*' Nanodrop said 29ng/µl after PCR Purification, which was strangely low. So used 16µl as recommended (in order to not exceed 0.2µg) but maximise amount of DNA.
 +
 +
*Gel electrophoresis: Run gel with following quantities:
 +
{|class="wikitable"
 +
|-
 +
|
 +
|pBAD
 +
|LC Luc (1)
 +
|-
 +
|DNA
 +
|17
 +
|17
 +
|-
 +
|6x Orange LD
 +
|3
 +
|3
 +
|}
µl°C
µl°C

Revision as of 17:01, 1 October 2010

Week 11: Monday 20th September - Sunday 26th September

Contents

Monday

Tuesday

107. Expt: Biobrick assembly of pBAD+Luc (PP + LC) and (Emily)

  • Plasmids already extracted
  • Restriction digest following protocol on p88 using these quantities (in µl):
pBAD PP Luc (1) PP Luc (2) LC Luc (1)
Nuclease-free H20 14 14 14 14
10x FD Buffer 2 2 2 2
Plasmid DNA 2 2 2 2
FD EcoRI 1 1 1 1
FD SpeI 1 0 0 0
FD XbaI 0 1 1 1
  • Gel electrophoresis
    • pBAD failed - band at ~5000bp, should be 1200
    • others look about right ~ between 3 & 5kb (should be 3600bp)
  • Gel extraction - results in -20°C freezer

Wednesday

108. Expt: Send off PP + pSB1C3, EPIC pBAD for sequencing (Emily and Peter)

  • Miniprep EPIC pBAD and nanodrop
  • Prepare correct concentrations (100ng/µl for plasmid, 3.2pmol/µl for primers) to be sent off

109. Expt: Biobrick assembly of pBAD + PP Luc (1), PP Luc (2), LC Luc (1), PP+pSB1C3

  • Peter 'miniprepped' PP+pSB1C3 to extract plasmid
  • pBAD was amplified after trying to grow overnight culture failed. The following protocol was used:

Add to a PCR tube:

10µl 2x Phusion Master Mix
1µl VF2 Primer
1µl VR Primer
8µl HPLC H20
20µl
  • Use stab to extract colony from pBAD plate and swirl in tube. Run following program on PCR machine saved as 'pBAD Amplification':
    • Heated lid at 110°C
    • Denaturation for 1m30s at 98°C
    • Cycle 35 times
      • Denaturation for 30s at 98°C
      • Elongation for 2m at 72°C
    • Elongation for 7m30s at 72°C
  • PCR Purification of pBAD from PCR reaction was performed using Qiagen kit
  • Restriction: repeating restriction of LC Luc (1) because tube from yesterday was dropped. Use protocol on p88 with following quantities (in µl):
pBAD LC Luc (1)
HPLC H20 0 14
10x FD Buffer 2 2
Plasmid DNA 16 (PCR product)* 2
FD EcoRI 1 1
FD XbaI 0 1
FD SpeI 1 0

'*' Nanodrop said 29ng/µl after PCR Purification, which was strangely low. So used 16µl as recommended (in order to not exceed 0.2µg) but maximise amount of DNA.

  • Gel electrophoresis: Run gel with following quantities:
pBAD LC Luc (1)
DNA 17 17
6x Orange LD 3 3

µl°C

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