906
edits
Changes
updated protocol after successful cloning
===Cloning===
* First digest pX335 vector by adding to a PCR tube(or use a frozen, pre-digested vector):** 1 2 ug of pX335,
** 1uL of 10X NEB Buffer 2.1
** 1 uL of CIAP
** 1 uL of BbsI
** water up to 10 uL
** Digest for 1h in, gel purify the fragment from an agarose gel, using the QIAEX II Kit and check the concentration by nanodrop.
* Next prepare the insert by annealing and phosphorylating the primers in a PCR tube:
** 4.5 uL of water** 1 uL of a 100 uM stock of each oligo with 4.5 uL of water** 2.5 uL of 5X ligase buffer
** 1 uL of T4 PNK
** Incubate at 37C for 30 mins then 95C for 5 mins to heat inactivate PNK then ramp down to 25C at 5C/min to allow the oligos to anneal, leave at room temperature** Dilute the annealed oligos 250X in water (2 uL + 498 uL of water)
* Combine the ligation mixture in an eppendorf tube:
** 5 50 ng of vector (~10 pmoles of vector)** 3 uL of annealed insert (~30 pmoles of Insert) or '''water as a blank'''** 2.5 uL of 4X 5X ligation buffer
** 1 uL of T4 DNA Ligase
** Water to 10 uL
* Incubate for 10 min at RT
* Transform 2uL of this into competent cells (see [[Transformation of Bacteria]]), plating the entire transformation.
