Difference between revisions of "Ligation and Transformation"
From GcatWiki
| Line 1: | Line 1: | ||
| − | '''Ligation | + | '''Ligation''' |
| − | #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 | + | #Dilute vector to 20 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 3 = ng insert, eg. 10ng x (700 bp/2100 bp) x 3 = 10 ng | 10 ng vector x (insert size/vector size) x 3 = ng insert, eg. 10ng x (700 bp/2100 bp) x 3 = 10 ng | ||
| − | #Combine vector, insert, and pure dH2O in a total volume of | + | # Combine vector, insert, and pure dH2O in a total volume of 8 ul in a 1.5 ml tube |
| − | + | # Add 1 ul 10X Ligation buffer and mix with pipetting. | |
| − | #Add | + | # Add 0.5 ul [http://www.neb.com/nebecomm/products/productM2200.asp Quick T4 DNA Ligase]and mix with pipetting. |
| − | #Add 0.5 ul [http://www.neb.com/nebecomm/products/productM2200.asp Quick T4 DNA Ligase] | + | # Add 0.5 ul of the appropriate restriction enzyme. Your total volume in the tube should now be 10 uL. |
| − | #Let stand 5 min at room temperature | + | #Let stand 5 min at room temperature. |
| − | #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 ligation tubes on ice for 2 minutes | + | #Place ligation tubes on ice for 2 minutes. |
| − | #Add | + | |
| − | #Let stand on ice for 5 minutes | + | '''Transformation''' |
| − | #Spread onto LB agar plates with the appropriate antibiotic | + | |
| + | #Dilute competent cells using dilution buffer. Add 70 uL of buffer if you are doing two plates. | ||
| + | #Add half of your competent cell/buffer mix, 60 uL in the case of two plates, to your ligation. | ||
| + | #Let stand on ice for 5 minutes. | ||
| + | #Spread onto LB agar plates with the appropriate antibiotic and mark the plates appropriately and legibly. | ||
| + | #Place your plates into the incubator. | ||
Revision as of 21:33, 2 February 2017
Ligation
- Plan to do a ligation with the vector only and one with vector + insert.
- Dilute vector to 20 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 3 = ng insert, eg. 10ng x (700 bp/2100 bp) x 3 = 10 ng
- Combine vector, insert, and pure dH2O in a total volume of 8 ul in a 1.5 ml tube
- Add 1 ul 10X Ligation buffer and mix with pipetting.
- Add 0.5 ul Quick T4 DNA Ligaseand mix with pipetting.
- Add 0.5 ul of the appropriate restriction enzyme. Your total volume in the tube should now be 10 uL.
- 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.
Transformation
- Dilute competent cells using dilution buffer. Add 70 uL of buffer if you are doing two plates.
- Add half of your competent cell/buffer mix, 60 uL in the case of two plates, to your ligation.
- Let stand on ice for 5 minutes.
- Spread onto LB agar plates with the appropriate antibiotic and mark the plates appropriately and legibly.
- Place your plates into the incubator.
