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#135130 PUAST sub - cloning problem

Posted by il0postino on 28 May 2012 - 08:48 AM in Molecular Biology

Vincent,

So you work with flies!

thought to just give you a suggestion protocol:

start with 3 ug of DNA of vector plasmid and 4.5 ug of insert plasmid. Digest each with EcoRI and NotI using NEB buff3 in ~100-150 ul total volume (with a bit of volume you dilute out possible impurities in the mini prep) use 2.5 ul of each enzyme (~10x overdigetion)
After 1-1.5 hours add CIP (calf intestine alkaline phosphatase) to the vector tube only (~2units) mix well and incubate both tubes for 30-45 minutes longer (also at 37C). This will reduce the background (also when using 2 different enzymes). Add gel loading buffer and load the two samples directly on a gel using multiple slots for each sample -because of the volume and to prevent overloading of the gel (for 9 kb use a gel below 1% agarose). Cut out the vector and insert bands carefully (if it is still overloaded just take the front of the band, also, use a long wave length UV box eg 360nm -DNA irradiated with short wave length UV will not clone).
Isolate the DNA from the gel slices and resuspend/collect in a small volume.
Ligate using 1.5 ul of each (will be appr. 1:3 molar ratio) in a total volume of 10ul -using 1 ul of 10x lig buffer 5.25 h2o and 0.75 T4 ligase. ligate o/n at 16C. Also take along controls: one using 1.5 h20 instead of vector and one using 1.5 h2o instead of insert.
Transform the most competent bacteria you have (2.5 ul lig in 50ul cells) and plate 2 dilutions of each transformation (to ensure single colonies on at least one plate).
Determine if the ligation shows enrichment as compared to the control plates.
If it looks good: Pick ~6 free colonies (pick colonies of all sizes present on the plate)
analyze and celebrate (positive thinking helps too!)




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