Difference between revisions of "Ligation and Transformation"
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| − | '''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 | + | 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 | + | # 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 on ice for | + | #Place ligation tubes on ice for 2 minutes. |
| − | #Add competent | + | |
| − | #Spread onto LB agar plates with the appropriate antibiotic | + | '''Transformation''' |
| + | |||
| + | #Before doing the actual transformation procedure, get your plates that you intend to use and place them in the incubator, agar side down so that they can warm up while getting the bacteria prepped. | ||
| + | #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 your mix onto LB agar plates with the appropriate antibiotic and mark the plates appropriately and legibly. | ||
| + | #Place your plates into the incubator. | ||
Latest revision as of 21:39, 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
- Before doing the actual transformation procedure, get your plates that you intend to use and place them in the incubator, agar side down so that they can warm up while getting the bacteria prepped.
- 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 your mix onto LB agar plates with the appropriate antibiotic and mark the plates appropriately and legibly.
- Place your plates into the incubator.
