Difference between revisions of "Ligation and Transformation"

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#Dilute vector to 10 ng/ul for use in the ligation
 
#Dilute vector to 10 ng/ul for use in the ligation
 
#Calculate the amount of insert needed with the following formula
 
#Calculate the amount of insert needed with the following formula
−
   10 ng vector x (insert size/vector size) x 6 = ng insert
+
   10 ng vector x (insert size/vector size) x 6 = ng insert, eg. 10ng x (700 bp/2100 bp) x 6 = 20 ng
−
#
+
#Combine vector, insert, and pure dH2O as a total volume of 5 ul in a 1.5 ml tube
−
#
+
#Heat at 42 C for 2 minutes
−
#
+
#Add 5 ul 2X Quick Ligation buffer and mix with pipetting
−
 
+
#Add 0.5 ul [http://www.neb.com/nebecomm/products/productM2200.asp Quick T4 DNA Ligase](and mix with pipetting
−
Go to the [http://partsregistry.org/Main_Page Registry ]and choose "Add a part"
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#Let stand 5 min at room temperature
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# Decide whether the new part is a '''Basic Part''' (discrete function units that cannot be subdivided, like promoter, RBS, coding sequence), '''Composite Part''' (functional unit composed of basic parts, like gene expression cassette, inverter), or '''Construction Intermediate''' (no function, byproduct of construction).
+
#Thaw competent cells on ice (eg. 100 ul of [http://www.zymoresearch.com/content/z-competent-e-coli-t3003-t3005-t3007-t3009-t3011-t3013-t3015-t3017 Z-competent JM109])
−
#Choose the number for the next available part
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#Heat ligation at 65 C for 10 minutes
−
#Be sure to include short and long descriptions of the part
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#Place on ice for 1 minute
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#Once data is available on the part, go back and include it as "Experience"
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#Add competent cells to ligation (at least 20 ul)and wait 5 minutes
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<br>
+
#Spread onto LB agar plates with the appropriate antibiotic

Revision as of 17:03, 7 June 2010

Ligation and Transformation

  1. Plan to do a ligation with the vector only and one with vector + insert
  2. Dilute vector to 10 ng/ul for use in the ligation
  3. Calculate the amount of insert needed with the following formula
  10 ng vector x (insert size/vector size) x 6 = ng insert, eg. 10ng x (700 bp/2100 bp) x 6 = 20 ng
  1. Combine vector, insert, and pure dH2O as a total volume of 5 ul in a 1.5 ml tube
  2. Heat at 42 C for 2 minutes
  3. Add 5 ul 2X Quick Ligation buffer and mix with pipetting
  4. Add 0.5 ul Quick T4 DNA Ligase(and mix with pipetting
  5. Let stand 5 min at room temperature
  6. Thaw competent cells on ice (eg. 100 ul of Z-competent JM109)
  7. Heat ligation at 65 C for 10 minutes
  8. Place on ice for 1 minute
  9. Add competent cells to ligation (at least 20 ul)and wait 5 minutes
  10. Spread onto LB agar plates with the appropriate antibiotic
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