Difference between revisions of "Restriction Enzyme Based Cloning"
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#If insert is PCR, run PCR then PCR purify (Qiagen kit). Elute in 30 uL EB | #If insert is PCR, run PCR then PCR purify (Qiagen kit). Elute in 30 uL EB | ||
#Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C. Add 1 uL of CIAP to the vector for the last ~10min at 37C. | #Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C. Add 1 uL of CIAP to the vector for the last ~10min at 37C. | ||
| − | #Gel purify both vector and insert (Qiagen kit). | + | #Gel purify both vector and insert (Qiagen kit). |
| − | #Combine | + | :*Run out on gel (see [[Preparing an Agarose Gel]]) |
| − | #Add 2 uL ligase buffer (single use aliquots) | + | :*On a gel box cut out appropriate bands and place in clean eppendorf tubes. Try to limit the exposure to UV light |
| − | #Incubate | + | :*Weigh out gel piece. For each mg add 3 uL of QG (orange) buffer. For example, for a 0.1g piece add 300 uL buffer. |
| − | #Transform 5-10 uL into 50 uL | + | :*Place at 55-65C until gel is dissolved (5-10 min) |
| + | :*Add to pink purification column, wash and elute in 30 uL | ||
| + | #Combine 6 uL insert with 1 uL vector. Place at 50-65C for 5-10 min to help sticky end binding. | ||
| + | #Add 2 uL ligase buffer (single use aliquots). | ||
| + | #Add 1 uL T4 DNA Ligase (Invitrogen). | ||
| + | #Incubate 2h-O/N at 16C (water bath in cold room). | ||
| + | #Transform 5-10 uL into 50 uL supercompetent cells: | ||
##Make 50 uL aliquots | ##Make 50 uL aliquots | ||
##Add DNA and mix gently | ##Add DNA and mix gently | ||
##Incubate on ice for 30min | ##Incubate on ice for 30min | ||
| − | ##Heat shock for | + | ##Heat shock for 45s at 42C |
##Place on ice for 2min | ##Place on ice for 2min | ||
##Add 450 uL LB | ##Add 450 uL LB | ||
##Incubate at 37C for 1h with shaking | ##Incubate at 37C for 1h with shaking | ||
| − | ##Plate 500 uL and grow O/N at 37C on appropriate plates | + | ##Plate 500 uL and grow O/N at 37C on appropriate antibiotic plates |
#Pick several colonies and digest to verify insert. Sequence if necessary. | #Pick several colonies and digest to verify insert. Sequence if necessary. | ||
Latest revision as of 14:40, 7 May 2009
- If insert is PCR, run PCR then PCR purify (Qiagen kit). Elute in 30 uL EB
- Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C. Add 1 uL of CIAP to the vector for the last ~10min at 37C.
- Gel purify both vector and insert (Qiagen kit).
- Run out on gel (see Preparing an Agarose Gel)
- On a gel box cut out appropriate bands and place in clean eppendorf tubes. Try to limit the exposure to UV light
- Weigh out gel piece. For each mg add 3 uL of QG (orange) buffer. For example, for a 0.1g piece add 300 uL buffer.
- Place at 55-65C until gel is dissolved (5-10 min)
- Add to pink purification column, wash and elute in 30 uL
- Combine 6 uL insert with 1 uL vector. Place at 50-65C for 5-10 min to help sticky end binding.
- Add 2 uL ligase buffer (single use aliquots).
- Add 1 uL T4 DNA Ligase (Invitrogen).
- Incubate 2h-O/N at 16C (water bath in cold room).
- Transform 5-10 uL into 50 uL supercompetent cells:
- Make 50 uL aliquots
- Add DNA and mix gently
- Incubate on ice for 30min
- Heat shock for 45s at 42C
- Place on ice for 2min
- Add 450 uL LB
- Incubate at 37C for 1h with shaking
- Plate 500 uL and grow O/N at 37C on appropriate antibiotic plates
- Pick several colonies and digest to verify insert. Sequence if necessary.
