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| {{Template:20.109}} | | {{Template:20.109}} |
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− | ==Introduction== | + | ==Ligations and Transformations== |
− | Today you will ligate your linearized M13K07 backbone with your epitope insert by mixing the two in the presence of ATP and an enzyme, T4 DNA ligase. During the ligation reactions, hydrogen bonds will form between the overhangs on the fragments, and then the ligase will repair the phosphate backbone, creating a stable circular plasmid.
| + | --[http://openwetware.org/wiki/User:Bryanh |Bryanh] 17:17, 16 February 2007 (EST) |
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− | [[Image:Macintosh HD-Users-nkuldell-Desktop-20109Ligation.png|thumb|left|300px| '''DNA ligation''']]
| + | ==Purpose== |
| + | ligate linearized M13K07 backbone with the epitope insert. transform ligated plasmid into bacteria and screen with Kan. |
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− | Hopefully, your ligation reactions will generate your desired construct, namely the M13K07 backbone carrying a myc-tag in either p8 or p3. Alternative ligation products may arise, including a simple reclosing of the singly cut M13K07 backbone. You can distinguish these reclosures from the correct constructs in a later step by looking at the PstI or BamHI sites, since these will be absent from the ligation products you are hoping to make.
| + | ==Protocols== |
| + | When you have a break from the work described below, be sure to examine the plaques you plated last time. Record the number of plaques on each plate, their appearance and any observations or conclusions you can draw. |
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− | You will transform today's ligation reactions into bacteria. During “transformation,” a single plasmid from the ligation mixture enters a single bacterium and, once inside, replicates and expresses the genes it encodes. One of the genes on the M13K07 genome leads to kanamycin-resistance. Thus, a transformed bacterium will grow on agar medium containing kanamycin. Untransformed cells will die before they can form a colony on the agar surface.
| + | ===Part 0=== |
| + | '''counting PFUs:''' |
| + | |
| + | ==Phage infection== |
| + | {| border="1" cellpadding="2" |
| + | !width="10"|A |
| + | !width="10"|B |
| + | !width="10"|C |
| + | |- |
| + | |'''Phage dilution'''||'''Phage Strain'''||'''PFUs Counted'''|| |
| + | |- |
| + | |none||E4||lawn|| |
| + | |- |
| + | |none||K07||lawn|| |
| + | |- |
| + | |10^-4||E4||too many to count|| |
| + | |- |
| + | |10^-4||K07||too many to count|| |
| + | |- |
| + | |10^-6||E4||2000|| |
| + | |- |
| + | |10^-6||K07||no PFUs (this is probably an error in our dilution step)|| |
| + | |- |
| + | |} |
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− | Most bacteria do not usually exist in a “transformation ready” state, but the bacteria can be made permeable to the plasmid DNA, and cells that are capable of transformation are referred to as “competent.” Competent cells are extremely fragile and should be handled gently, specifically kept cold and not vortexed. The transformation procedure is efficient enough for most lab purposes, with efficiencies as high as 10^9 transformed cells per microgram of DNA, but it is important to realize that even with high efficiency cells only 1 DNA molecule in about 10,000 is successfully transformed.
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− | [[Image:Be109transformation.jpg|thumb|left|200px|'''Bacterial transformation''']]
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− | <br style="clear:both" />
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− | ==Protocols==
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− | When you have a break from the work described below, be sure to examine the plaques you plated last time. Record the number of plaques on each plate, their appearance and any observations or conclusions you can draw.
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| ===Part 1: Ligation reactions=== | | ===Part 1: Ligation reactions=== |
− | For your ligation, you will mix the M13K07 backbone you prepared with the annealed oligonucleotides. As control reactions you will also prepare a “bkb, no ligase” reaction to control for any errant uncut plasmid that might have wandered into your solutions. Additionally you will prepare “bkb only, plus ligase” reaction to assess the frequency of backbone religation. | + | For your ligation, you will mix the M13K07 backbone you prepared with the annealed oligonucleotides. As control reactions you will also prepare a |