Team:Newcastle/22 June 2010

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'''Tuesday'''
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{{Team:Newcastle/mainbanner}}
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===Qiagen miniprep: Plasmid extraction===
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The 5ml culture set up yesterday was pelleted down using the microcentrifuge, the pellet was resuspended in buffer P1.
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Lysis buffer P2 was used to release the DNA from the cell (this buffer contains RNAse to help reduce RNA contamination). PE buffer containing ethanol was used as a wash. We lysed the cells for 1 minute, gently inverting the tube. We neutalised  the lysis with N3 buffer and centrifuged for 10minutes, only the '''plasmid DNA was left in suspension'''. The DNA was eluted at low salt concentration through a column membrane.
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===Digest===
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==Aims==
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The aim of today's Lab practice was to extract the plasmids for GFP and RFP, digest the 2 plasmids and extract the 2 inserts and 1 of the backbones (vector)from an agarose gel. From this a ligation was set up. 
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Cut with restriction enzyme ''EcoR1'' and ''Pst1'' 1 μl of each.
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==Materials and Protocol==
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10*buffer ... so 3 μl in 30μ1 therefore we can have 25μ1 of DNA
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Also no more than 10% Glycerol in the enzyme solution or the reaction would be inhibited.
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===Gel extraction===
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===QIagen Miniprep Kit Using a Microcentrifuge for Plasmid Extraction===
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Please refer to: [[Team:Newcastle/Qiagen Minipreps|Qiagen Minipreps]] for materials required and protocol.
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====Agarose gel====
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===Digest===
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Please refer to: [[Team:Newcastle/Restriction digests| Restriction digests]] for materials required and protocol.
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Agarose gel is used for DNA seperation. 1% agarose is used because it is suitable for most kilobase pairs of DNA. We used 60ml for our gel. We used SafeView dye to bind the DNA so that the DNA is visible. We used TAE buffer and boiled the mixture in a microwave to melt it. We then let it cool and poured it into the tank to set.
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===Gel extraction===
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Please refer to: [[Team:Newcastle/Gel extraction| Gel extraction]] for materials required and protocol.
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===Agarose Gel Electrophoresis===
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Please refer to: [[Team:Newcastle/Gel electrophoresis| Gel electrophoresis]] for materials required and protocol.
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====Electrophoresis====
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{|
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|-
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In lane one we loaded the molecular marker 5μl. In lane three we loaded the digested RFP plasmid. In lane five we loaded the digested GFP plasmid. In lane eight we loaded 5μl RFP plasmid and 1μl sample buffer. Lanes two, four, six and seven are empty.  
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|[[Image:Newcastle_lab_1.jpeg|thumb|Dr Wendy demostrating how to use gel electrophoresis machine]]
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|[[Image:Newcastle_lab_2.jpeg|thumb|Gel tank]]
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Pictures of equipments/gel:
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|[[Image:Newcastle_lab_3.jpeg|thumb|Deena pouring hot molten gel]]
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|-
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|}
====Cut gel out====
====Cut gel out====
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Dark room pictures of gel
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{|
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|-
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|[[Image:Newcastle_lab_4.jpeg|thumb|Putting the gel on the geldoc]]
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|[[Image:Newcastle_lab_5.jpeg|thumb|Preparing to cut the gel]]
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|[[Image:Newcastle_lab_6.jpeg|thumb|Gel illuminated by the UV light which is emitted by the geldoc]]
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|-
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|}
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We cut the inserts which were about 900bp and we use the backbone from the RFP plasmid.
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We cut the inserts which were about 900bp and we use the backbone from the plasmid consisting ''rfp''.
====QIAquick Gel extraction kit====
====QIAquick Gel extraction kit====
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Phil with scalpel in dark room.#
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{|
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|-
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|[[Image:Newcastle_lab_7.jpeg|thumb|Phil cutting the gel]]
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|[[Image:Newcastle_lab_8.jpeg|thumb|Transferring the cut gel part into a 2 ml microfuge tube]]
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|-
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|}
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==Set up ligation==
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{|border=1
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|-
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!Reagents
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!1:3(μl)
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!1:5(μl)
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!Vector(μl)
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|-
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|V
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|0.8
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|0.8
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|1
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|-
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|G
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|2.7
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|4
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|
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|-
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|R
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|5.4
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|7.7
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|
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|-
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|LT4
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|1
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|1
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|1
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|-
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|LB
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|1.1 
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|1.5
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|1
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|-
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|H2O
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|0
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|0
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|7
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|-
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|Total Volume
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|11.0
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|15.0
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|10.0
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|}
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==Transformation protocol==
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Please refer to: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']] for materials required and protocol.
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==Nanodrop Protocol==
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Please refer to: [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for materials required and protocol.
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The DNA fragments are cut from the agarose gel with a scalpel. Three parts of QG buffer is added to one part of gel and the mixtures are dissolved at the temperature of 50°C. The tubes are vortexed every 2-3 minutes to help dissolve the gel. 1 part of isopropanol is added to the sample and mix. The microcubes were spinned in the microfuge for 1 minute.
 
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====Weighing====
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{|
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|-
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|[[Image:Newcastle_nanodrop_1.jpeg|thumb|A typical curve to be achieved from a nanodrop spectrophotmeter]]
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|[[Image:Newcastle_nanodrop_2.jpeg|thumb|Phil analyzing a sample on the nanodrop machine]]
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|[[Image:Newcastle_nanodrop_3.jpeg|thumb|Analyzed data on the screen]]
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|-
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|}
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===Set up ligation===
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==Outcome==
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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="2" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
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!align="center"|[[Team:Newcastle|Home]]
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!align="center"|[[Team:Newcastle/Team|Team]]
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!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Newcastle Official Team Profile]
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!align="center"|[[Team:Newcastle/Project|Project]]
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!align="center"|[[Team:Newcastle/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:Newcastle/Modelling|Modelling]]
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!align="center"|[[Team:Newcastle/Notebook|Notebook]]
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!align="center"|[[Team:Newcastle/Safety|Safety]]
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|}
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===Nanodrop===
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{{Team:Newcastle/footer}}

Latest revision as of 20:42, 21 October 2010

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Contents

Aims

The aim of today's Lab practice was to extract the plasmids for GFP and RFP, digest the 2 plasmids and extract the 2 inserts and 1 of the backbones (vector)from an agarose gel. From this a ligation was set up.

Materials and Protocol

QIagen Miniprep Kit Using a Microcentrifuge for Plasmid Extraction

Please refer to: Qiagen Minipreps for materials required and protocol.

Digest

Please refer to: Restriction digests for materials required and protocol.

Gel extraction

Please refer to: Gel extraction for materials required and protocol.

Agarose Gel Electrophoresis

Please refer to: Gel electrophoresis for materials required and protocol.

Dr Wendy demostrating how to use gel electrophoresis machine
Gel tank
Deena pouring hot molten gel

Cut gel out

Putting the gel on the geldoc
Preparing to cut the gel
Gel illuminated by the UV light which is emitted by the geldoc

We cut the inserts which were about 900bp and we use the backbone from the plasmid consisting rfp.

QIAquick Gel extraction kit

Phil cutting the gel
Transferring the cut gel part into a 2 ml microfuge tube

Set up ligation

Reagents 1:3(μl) 1:5(μl) Vector(μl)
V 0.8 0.8 1
G 2.7 4
R 5.4 7.7
LT4 1 1 1
LB 1.1 1.5 1
H2O 0 0 7
Total Volume 11.0 15.0 10.0

Transformation protocol

Please refer to: Transformation of E. coli for materials required and protocol.

Nanodrop Protocol

Please refer to: Nanodrop Spectrophotometer for materials required and protocol.


A typical curve to be achieved from a nanodrop spectrophotmeter
Phil analyzing a sample on the nanodrop machine
Analyzed data on the screen

Outcome

Home Team Official Team Profile Project Parts Submitted to the Registry Modelling Notebook Safety
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