Change Backbone

This protocol walks you through how to essentially copy-paste a gene from one vector backbone into another.

Run a gel w/ 5% PCR product to check correct amplicon size
1% agarose gel (in 1X TAE) with 3uL EtBr per 100mL –pour thin, use 5uL 1Kb+ ladder (for little well in a minigel) Save 50% of the remaining PCR product, freeze at -20

Clean up the PCR product
transfer to different buffer for RE digest Take remaining 50% PCR product, add 5X volume Buffer PB Transfer to purple tube from gel extraction kit (pcr cleanup kit) Spin, aspirate 750uL PE Spin, aspirate, spin to dry Elute with 50uL EB Save 50% product

RE digest
cut off ends so can put into correct vector Cut inserts and vector with correct restriction enzymes High efficiency enzymes! (enxonuclease chewing which will decrease ligation efficiency) Want 30uL reaction total (from above, using 25uL product)

0.5uL enzyme A / reaction + 0.5uL enzyme B/ reaction

Buffer at 10X, 3uL

25uL sample

1uL water

Cut for 2 hours at 37C

Run gel to separate insert from ends
30uL RE digest + 6uL bromo blue (or glycerol) 1% agarose gel (thin) + EtBr in gel ~45mins @ 120V

Isolate Insert
Cut bands from gel (razor, saran wrap, labeled tubes, UV box) Melt gel/isolate dna Re-suspend gel in at least 3 gel volumes of QG buffer (max = 750uL / tube …550 uL added) Melt at 55C (waterbath) for 5 mins

Make sure no gel pieces left Load into gel extraction column (purple) Spin, asp 750uL PE Spin, asp Spin Elute w/ warm EB, 5 mins

Want inserts more concentrated (elute in 25uL), plasmid more dilute (60uL) Spin 1 min

Run quant gel
1% agarose, thin gel, with EtBr in it… Want 3 molar excess of plasmid Run different volumes of insert/plasmid to get an idea of relative quants: 10uL, 2uL plasmid; 5uL each pcr product….

Bench top ligation
Care for ligase buffer:

Thaw slowly and fully at 37C, vortex etc

Vortex, spin, mix before use

Make sure can smell DTT (if not, it’s dead)

Make sure close really tightly before storing again Care for enzymes: never vortex, keep very frozen, re-store immediately. 0.5uL plasmid per reaction (based on gel “quant”)—want large excess of plasmid! (variable) 5uL pcr product (variable) 5uL 2X Ligase mix:

1uL 10X ligase buffer (must be warmed 20 mins before rxn)

0.5uL T4 ligase (in cloning box)

3.5uL H2O Add plasmid and annealed product first, then ligase mix Mix! Set at RT for 1-2hrs (60min minimum). Longer = more colonies

Finish cloning
Transform bacteria, plate, grow, select colonies, grow, miniprep, sequence, use!