Team:UNICAMP-EMSE Brazil/protocols/Ligation

From 2011.igem.org

(Difference between revisions)
(Created page with "{{Template:UNICAMP-EMSE Brazil_Main_Menu}} ==Ligation== Quantificate the plasmid using absorbance at 260 nm and 280 nm. Concentration of plasmid (μg/mL) = A260*50*dilution ...")
Line 31: Line 31:
T4 DNA Ligase – 1 uL
T4 DNA Ligase – 1 uL
Vol final - 20ul
Vol final - 20ul
 +
 +
 +
<html>
 +
<head>
 +
<script language='JavaScript' type='text/JavaScript'>
 +
 +
function makeQuote() {
 +
 +
Q = new Array();
 +
 +
 +
Q[0] = "<i>I’m Luke Skywalker, I’m here to rescue you.</i>Luke, Star Wars: Episode IV-A New Hope (1977)"
 +
Q[1] = "<i>Watch your mouth kid, or you’ll find yourself floating home.</i>Star Wars: Episode IV-A New Hope (1977)"
 +
Q[2] = "<i>Evacuate in our moment of triumph? I think you overestimate their chances.</i> Star Wars: Episode IV-A New Hope (1977)"
 +
Q[3] = "<i>The Force is strong with this one.</i> Star Wars: Episode IV-A New Hope (1977)"
 +
Q[4] = "<i> I have you now!</i> Darth Vader, Star Wars: Episode IV-A New Hope (1977)"
 +
Q[5] = "<i> Fear is the path to the dark side. Fear leads to anger, anger leads to hate, hate leads to suffering.</i> MsC Yoda, Star Wars Episode I: The Phantom Menace (1999)"
 +
Q[6] = "<i> No blasters! No blasters!.</i> A bartender, Star Wars: Episode IV-A New Hope (1977)"
 +
Q[7] = "<i> Go to the center of the gravity's pull, and find your planet you will...</i> M.Sc. Yoda, Star Wars Episode II: Attack of the Clones (2002)"
 +
Q[8] = "<i> Clear, your mind must be if you are to discover the real villains behind the plot.</i> M.Sc. Yoda, Star Wars Episode II: Attack of the Clones (2002)"
 +
Q[9] = "<i> Much to learn, you still have.</i> M.Sc. Yoda, Star Wars Episode II: Attack of the Clones (2002)"
 +
Q[10] = "<i> Meditate on this, I will.</i> M.Sc. Yoda, Star Wars Episode II: Attack of the Clones (2002)"
 +
Q[11] = "<i> Use the Force, Luke.</i> M.Sc. Yoda, Star Wars Episode II: Attack of the Clones (2002)"
 +
Q[12] = "<i> May the Force be with you.</i> Mentioned several times throughout the Star Wars saga"
 +
Q[13] = "<i> I really need a Jedi Bacteria.</i> Iolanda Albuquerque, UNICAMP-EMSE team advisor"
 +
Q[14] = "<i> Another splendid day in Brazil!!!</i> Marc Emery, UNICAMP-EMSE team student"
 +
Q[15] = "<i> Dont' cut, let your hair volume expressing all their feelings...</i> Thibault Sabattier (UNICAMP-EMSE team student) about Nemailla's hair"
 +
Q[16] = "<i> What a good life!!</i> Louise Marais (UNICAMP-EMSE team student) about living in Brazil"
 +
Q[17] = "<i> Gosh!I will have nigthmares tonight! </i> Danieli Gonçalves (UNICAMP-EMSE team student) about Thibault wearing Marianna's dress"
 +
Q[18] = "<i> I will revenge myself against you and Marc Emery!!!</i> Nemailla Bonturi (UNICAMP-EMSE team advisor)"
 +
Q[19] = "<i> Yeah, those dresses deserve us!</i> Marc Emery (UNICAMP-EMSE team student) about Marianna's dresses"
 +
Q[20] = "<i> I am really starting getting worried with you guys!!</i> Iolanda Albuquerque (UNICAMP-EMSE team advisor)"
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
index = Math.floor(Math.random() * Q.length);
 +
 +
document.writeln(Q[index]);
 +
 +
}
 +
 +
</script>
 +
 +
</head>
 +
 +
<style>
 +
#newquote {color:gray; font-family:verdana; font-size:11px; text-align:right; padding:5px;}
 +
 +
#newquote i {display:block; color:black; font-family:georgia, trebuchet ms; font-size:13px;}
 +
 +
#newquote small {display:block; text-align:right; margin-top:10px;}
 +
 +
]]></b:skin>
 +
</style>
 +
</html>
 +
 +
 +
<html>
 +
<head>
 +
<div id='newquote'>
 +
 +
<script language="javascript">
 +
 +
makeQuote();
 +
 +
</script>
 +
</div>
 +
</head>
 +
</html>
 +
 +
 +
 +
 +
 +
 +
<html>
 +
<head>
 +
<script type="text/javascript">
 +
 +
  var _gaq = _gaq || [];
 +
  _gaq.push(['_setAccount', 'UA-13209770-10']);
 +
  _gaq.push(['_trackPageview']);
 +
 +
  (function() {
 +
    var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;
 +
    ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';
 +
    var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);
 +
  })();
 +
 +
</script>
 +
</head>
 +
</html>

Revision as of 00:28, 23 August 2011

Home Project Methods Results Data Team Notebook Human Practices Safety Profile Sponsors Wix

Ligation

Quantificate the plasmid using absorbance at 260 nm and 280 nm. Concentration of plasmid (μg/mL) = A260*50*dilution Ratio 260/280 nm should be between 1,8-2,0.

Use this online tool to calculate how much insert to calculate how much insert we will need to use in the reaction (http://www.promega.com/techserv/tools/biomath/calc06.htm).

Or

[length of insert (kb)/ length of vector (kb)]* ng of vector = ng of insert for a 1:1 ratio


It´s better to use at least 100 ng of vector and a reaction ratio of 3:1 (insert:vector)

Example:

Length of insert (in kb) = 0.7 Amount of vector (in ng) = 100 Length of vector (in kb) = 5

You need 14 ng de insert for a 1:1 molar ratio For the 3:1= 42 ng de inserto.

Ligation reaction (using invitrogen ligase)

5x T4 DNA Ligase - 4uL Vetor – 42 ng Inserto – 100 ng T4 DNA Ligase – 1 uL Vol final - 20ul






Retrieved from "http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/protocols/Ligation"