Restriction Enzyme Based Cloning: Difference between revisions
updated with gel purification |
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#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). | ||
:*Run out on gel (see [[Preparing an Agarose Gel]] | :*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 | :*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. | :*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. | ||