Difference between revisions of "Ligation and Transformation"
From GcatWiki
| Line 2: | Line 2: | ||
#Plan to do a ligation with the vector only and one with vector + insert | #Plan to do a ligation with the vector only and one with vector + insert | ||
| − | #Dilute vector to 10 ng/ul for use in the ligation | + | #Dilute vector to 10 ng/ul for use in the ligation, use Millipore pure dH2O |
#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, eg. 10ng x (700 bp/2100 bp) x 6 = 20 ng | 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 | + | #Combine vector, insert, and pure dH2O in a total volume of 5 ul in a 1.5 ml tube |
#Heat at 42 C for 2 minutes | #Heat at 42 C for 2 minutes | ||
#Add 5 ul 2X Quick Ligation buffer and mix with pipetting | #Add 5 ul 2X Quick Ligation buffer and mix with pipetting | ||
| Line 12: | Line 12: | ||
#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]) | #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]) | ||
#Heat ligation at 65 C for 10 minutes | #Heat ligation at 65 C for 10 minutes | ||
| − | #Place on ice for | + | #Place ligation tubes on ice for 2 minutes |
| − | #Add competent cells to ligation | + | #Add at least 20 ul of competent cells to ligation using a cold pipette tip |
| + | #Let stand on ice for 5 minutes | ||
#Spread onto LB agar plates with the appropriate antibiotic | #Spread onto LB agar plates with the appropriate antibiotic | ||
Revision as of 13:51, 16 June 2010
Ligation and Transformation
- Plan to do a ligation with the vector only and one with vector + insert
- Dilute vector to 10 ng/ul for use in the ligation, use Millipore pure dH2O
- 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
- Combine vector, insert, and pure dH2O in 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 Quick T4 DNA Ligase(and mix with pipetting
- Let stand 5 min at room temperature
- Thaw competent cells on ice (eg. 100 ul of Z-competent JM109)
- Heat ligation at 65 C for 10 minutes
- Place ligation tubes on ice for 2 minutes
- Add at least 20 ul of competent cells to ligation using a cold pipette tip
- Let stand on ice for 5 minutes
- Spread onto LB agar plates with the appropriate antibiotic
