Team:UNICAMP-EMSE Brazil/protocols/Ligation

From 2011.igem.org

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Quantificate the plasmid using absorbance at 260 nm and 280 nm.  
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*Quantificate the plasmid using absorbance at 260 nm and 280 nm.  
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Concentration of plasmid (μg/mL) = A260*50*dilution
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*Concentration of plasmid (μg/mL) = A260*50*dilution
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Ratio 260/280 nm should be between 1,8-2,0.
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*Ratio 260/280 nm should be between 1,8-2,0.
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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).
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*Use [http://www.promega.com/techserv/tools/biomath/calc06.htm 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).
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Or  
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<font color=green> '''Or '''</font>
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[length of insert (kb)/ length of vector (kb)]* ng of vector  =  ng of insert for a 1:1 ratio
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*[length of insert (kb)/ length of vector (kb)]* ng of vector  =  ng of insert for a 1:1 ratio
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It´s better to use at least 100 ng of vector and a reaction ratio of 3:1 (insert:vector)
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''It´s better to use at least 100 ng of vector and a reaction ratio of 3:1 (insert:vector)''
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Example:
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'''Example:'''
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Length of insert (in kb) = 0.7
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:*Length of insert (in kb) = 0.7
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Amount of vector (in ng) = 100  
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:*Amount of vector (in ng) = 100  
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Length of vector (in kb) = 5
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:*Length of vector (in kb) = 5
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You need 14 ng de insert for a 1:1 molar ratio
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::*You need 14 ng de insert for a 1:1 molar ratio
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For the 3:1= 42 ng de inserto.
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::*For the 3:1= 42 ng de inserto.
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Ligation reaction (using invitrogen ligase)
 
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5x T4 DNA Ligase - 4uL
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===Ligation (protocol adapted from Fermentas)===
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Vetor – 42 ng
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Inserto – 100 ng
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*Plasmid (~50-60ng)
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T4 DNA Ligase – 1 uL
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*Insert DNA purified (the ratio was determined according to the fragment and plasmid sizes, ranging from 1:1 to 1:4)
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Vol final - 20ul
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*2μl of T4 DNA Ligase Buffer
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*1μl of T4 DNA Ligase
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*Sterile water
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::*Final volume = 20μl
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Add the reaction components to a sterile 0,2 sterile tube. Mix gently.
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Keep the tubes at 22˚C for 1 hour or overnight at 4˚C.
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Latest revision as of 15:20, 27 September 2011

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UNICAMP-EMSE Brazil Ligation.png

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 [http://www.promega.com/techserv/tools/biomath/calc06.htm 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 (protocol adapted from Fermentas)

  • Plasmid (~50-60ng)
  • Insert DNA purified (the ratio was determined according to the fragment and plasmid sizes, ranging from 1:1 to 1:4)
  • 2μl of T4 DNA Ligase Buffer
  • 1μl of T4 DNA Ligase
  • Sterile water
  • Final volume = 20μl

Add the reaction components to a sterile 0,2 sterile tube. Mix gently.

Keep the tubes at 22˚C for 1 hour or overnight at 4˚C.






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