Team:Imperial College London/Wetlab/Protocols/Cellculture

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=Cell culture protocols=
{| style="color:#CCC; background-color:#325d97;" cellpadding="6" cellspacing="0" border="3"
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! Assay
! Assay
! Overview and Aims
! Overview and Aims
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <b>PP1</b>: IPTG Toxicity
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| <b>CC1</b>: Miniprep
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| [[Team:Imperial_College_London/Wetlab/Protocols/IPTGgrowth |IPTG Toxicity]]
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| [[Team:Imperial_College_London/ |Link]]
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| To investigate the effect of our IPTG inducer on growth of our cultures.
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| * Harvest smaller amounts of genomic DNA<br>
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* Measure growth rates at 28 degrees Celsius on minimal growth media so that subsequent testing timings etc. can be streamlined
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* Determine the effect of IPTG toxicity on growth w/o any protein production complications <br>
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <b>PP3</b>: Cellulase
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| <b>CC2</b>: Cellulase
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| [[Team:Imperial_College_London/Wetlab/Protocols/Cellulase |Cellulase]]
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| [[Team:Imperial_College_London/ |Link]]
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| Aims <br>
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| * Harvest larger amount of genomic DNA<br>
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <b>PP4</b>: PAH
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| <b>CC3</b>: Cell competence
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| [[Team:Imperial_College_London/Wetlab/Protocols/PAH |PAH]]
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| [[Team:Imperial_College_London/ |Link]]
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| Aims<br>
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| * Making cells more competent to taking up DNA <br>
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|}
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <b>CC4</b>: Ligation
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| [[Team:Imperial_College_London/ |Link]]
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| * Ligation of different DNA constructs together<<br>
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==CC6: Transformation into Top10 using chemical competence==
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <b>CC5</b>: Transformation (BL21)
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| [[Team:Imperial_College_London/ |Link]]
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| * Transformation into BL21 cells using electrical shock <<br>
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===Protocol===
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <b>CC6</b>: Transformation (Top10)
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| [[Team:Imperial_College_London/Wetlab/Protocols/Cellculture/TransTop10 |Transformation into Top10]]
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| * Transformation into chemically competent Top10 cells <<br>
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1. Switch on water bath to 42 degrees. <br>
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <b>CC7</b>: Make LB plates
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| [[Team:Imperial_College_London/Wetlab/Protocols/Cellculture/LBPlates |Make LB plates]]
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| * Making plates of LB agar for cell culturing<br>
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2. Aliquot out LB media and place in water bath (roughly 1 ml for each sample)<br>
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <b>CC7</b>: Extract DNA from registry
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| [[Team:Imperial_College_London/Wetlab/Protocols/ExtractingDNA |Extracting DNA]]
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| * Extracting the required DNA from the registry<br>
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3. Chill DNA and 15ml round bottom tubes on ice (1 for each sample)<br>
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| <b>CC13</b>: Genome Prep for Restriction asay
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| [[Team:Imperial_College_London/Wetlab/Protocols/GenomePrep |Genome Prep]]
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| * Preperation of the genomic DNA for restriction digest assay.
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4. When all this is done, take out cells from -80 degrees freezer and thaw on ice.<br>
 
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5. When cells are thawed, add 30ul of cells to each round bottom tube.<br>
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|}
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6.  Add 3ul of registry DNA or 1ul of midi DNA to tube.  Swirl gently.
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7. S
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Latest revision as of 11:05, 30 September 2009

Cell culture protocols

Section Assay Overview and Aims
CC1: Miniprep Link * Harvest smaller amounts of genomic DNA
CC2: Cellulase Link * Harvest larger amount of genomic DNA
CC3: Cell competence Link * Making cells more competent to taking up DNA
CC4: Ligation Link * Ligation of different DNA constructs together<
CC5: Transformation (BL21) Link * Transformation into BL21 cells using electrical shock <
CC6: Transformation (Top10) Transformation into Top10 * Transformation into chemically competent Top10 cells <
CC7: Make LB plates Make LB plates * Making plates of LB agar for cell culturing
CC7: Extract DNA from registry Extracting DNA * Extracting the required DNA from the registry
CC13: Genome Prep for Restriction asay Genome Prep * Preperation of the genomic DNA for restriction digest assay.


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