• Thaw all frozen liquid completely (e.g. DNA stocks, miniprep DNA, ligation mix, digests, buffers, primers, etc.).  Then mix well by vortexing a few times & performing quick spins to collect all liquids at bottom of tubes before opening.
• Keep all reactions/buffers on ice after thawing.
• Quick spin tubes of enzymes before opening & usage.
• For digests & ligations, mix all ingredients well by swirling a pipet tip and pipetting up & down.
• For each tube: label name of DNA/plasmid/vector/PCR product + label name(s) of enzymes if digesting/digested with enzyme + label AP if treated with alkaline phosphatase.
• For each tube: label ligation if it is a ligation reaction with vector & insert names.
• For each tube: label DNA concentration after nanodrop.
• For each tube: label date.

PCR:

• Quick spin new tube of primers when they arrive to collect content at the bottom of tube.
  
• Add ddH20 to reconsitute primers.  Incubate 1 hour at RT.  Vortex vigorously.  Quick spin to collect all liquid.
• For 50 ul PCR reaction: use 50 ng template DNA + 10 ul 5X One Taq PCR reaction buffer + 2 ul 10 mM dNTPs + 5 ul sense primer + 5 ul anti-sense primer + water.
  
• Make sure all buffers & DNA are thawed completely (room temp is OK), then mix, vortex, quick spin to collect liquid at the botton of tube.  Then put everything on ice.
• Calculate the volume of template DNA needed from DNA/stock concentration then calculate how much water is needed to make 50 ul. 
  
• Template DNA concentration can be varied for optimization (20-100 ng). 
• Mix all ingredients well (by pipeting or vortex).
• Spin tube to remove bubbles.
  
• Add 1 ul One-Taq polymerase.
• Mix well by swirling/pipetting up & down many times.
• Quick spin to collect all liquids at the bottom of tubes.
  
• Optimize thermocycling parameters (denaturing temperature-depends on the template DNA, annealing temperature-depends on the length and composition of the primers, elongation time-depends on the length of the PCR product to be amplified) when necessary.
• Run 2 ul PCR reaction on TBE gel to check PCR efficiency (how strong is the PCR band/product) & specificity (whether the right size product is amplified).
• Scale up to 2X50 ul reactions if necessary.
• Freeze PCR reaction until blue filter purification.

Restriction Digest for PCR products:

• For a 100 ul digestion reaction: use entire 50 uL tube of blue filter purified DNA + 10 ul 10xNEB buffer + 1 ul 100xBSA + 39 uL water.
• Make sure all buffers & DNA are thawed completely (room temp is OK), then mix, vortex, quick spin to collect liquid at the botton of tube.  Then put everything on ice.
• Mix all ingredients well (by pipeting or vortex).
• Spin tube to remove bubbles.
  
• Add 5 ul of each enzyme per 100 ul digest reaction.
• Mix well by swirling/pipetting up & down many times.
• Quick spin to collect all liquids at the bottom of tubes.
  
• Incubate in 37 degree room overnight.
• Heat inactivate at 65 degrees for 15 min.
• Freeze until run agarose TAE gel.  General guideline: Use 0.8% agarose for >2kb.  Use 1% agarose for 0.5-2 kb. Use 1.2% for <0.5 kb

Restriction Digest for vector:

• For a 100 ul digestion reaction: use 20 ug plasmid + 10 ul 10xNEB buffer + 1 ul 100xBSA + water.
• Calculate the volume of plasmid DNA needed from DNA/stock concentration then calculate how much water is needed to make 100 ul. 
• Make sure all buffers & DNA are thawed completely (room temp is OK), then mix, vortex, quick spin to collect liquid at the botton of tube.  Then put everything on ice.
• Mix all ingredients well (by pipeting or vortex).
• Spin tube to remove bubbles.
  
• Add 5 ul of each enzyme per 100 ul digest reaction.
• Mix well by swirling/pipetting up & down many times.
• Quick spin to collect all liquids at the bottom of tubes.
  
• Incubate at 37 degrees/waterbath for 3 hours.
• Heat inactivate at 65 degrees for 15 min.
• Chill reaction tube on ice for 5 min - or until solution is cooled below 37 degrees.
• Add 12 ul 10X antartic phosphatase buffer.
• Add 5 ul antartic phosphatase.
• Mix well by swirling/pipetting up & down many times.
• Quick spin to collect all liquids at the bottom of tubes.
  
• Incubate at 37 degrees/waterbath for 1 hour.
• Heat inactivate at 65 degrees for 15 min.
• Freeze until run 1.2% Agarose 1xTAE gel or 1% Agarose 1XTBE gel.

• Use new blue filters for  gel slices. Use <250 mg gel slice per 1.25 ml QX1.
• Dissolve gel well in QX1 at 65 degrees (~30 min).  Mix by inverting every 5 min.
• After gel is dissolved.  Wait until solution is cooled to room temperature before applying to blue filter.
  
• Add 0.5 ml DNA/QX1 to filter upper well.
• Spin 2 min at 500 xg.
• Discard liquid from bottom tube.
• Repeat until all DNA/QX1 is loaded.
• Wash filter once with QX-1. Add 0.5 ml QX-1. Spin 1 min at 1000 xg.
• Wash filter 3 times with PE/ethanol.  Add 0.5 ml PE/ethanol.  Spin 1 min at 1000 xg.
• Change to new bottom tube.
• Spin 1 min at 15,000 xg.
• Change to another new bottom tube.
• Spin 2 min at 15,000 xg.
• Change to a new eppendorf tube with lid cut off.  Label tube with name of DNA, digest enzymes.
• Incubate filter with 10 mM Tris pH 8.8 at 37 degrees for 10 min.
• Spin 2 min at 15,000 xg.
• Nanodrop.
• Label concentration of DNA on tube.

• Make sure all buffers & DNA are thawed completely (room temp is OK), then mix, vortex, quick spin to collect liquid at the botton of tube.
• Then keep everything on ice.
• For a 20 ul ligation reaction: use 100 ng plasmid (gel purified after digest & AP) + 500 ng insert (gel purified after digest) + 2 ul of 10xT4 ligase buffer + water.
• Calculate the volumes of vector & insert DNA needed from DNA/stock concentrations then calculate how much water is needed to make 20 ul.  Keep everything on ice.
• Mix all ingredients well (by pipeting or vortex).
• Spin tube to remove bubbles.
• Add 1 ul T4 ligase.
  
• Mix well by swirling/pipetting up & down many times.
• Quick spin to collect all liquids at the bottom of tubes.
  
• Incubate at 16 degrees overnight.
• Freeze until transformation.

Transformation of ligation:

• Add 5 ul ligation to polypropylene tube on ice.
• Add 25-50 ul DH5alpha super competent bugs.
• Incubate on ice for 1 hour.
• Heat shock at 42 degrees 45 sec.
• Incubate in ice for 5 min.
• Add LB 1 ml.
• Shake at 37 degrees for 1 hour.
• Plate onto 2 LB/antibiotic plates.
• Label the bottom of plates (not the lids).
• Make sure there is not running liquid (open the lids & air dry the plates right-side -up until there is no running liquid)
  
• Put the plates up-side-down in 37 degree room.
  

• Pick single colonies from ligation/transformation.
• Incubate with 5ml LB/antibiotics at 37 degrees shake overnight.
• Spin 1.5-3 ml bugs down.  Remove supernatant.  Make sure no liquid is left.
• Add 5 ml LB/antibiotics to the rest of bacterial culture.
• Save the rest of bacterial culture at 4 degrees or RT for a few days.  
• Miniprep or freeze until miniprep.

Miniprep:

• Add 300 ul Solution I to resuspend bacterial pellet.
• Mix well by vortexing.
• Add 300 ul Solution II to lyze cells.
  
• Mix gently by inverting 10 times.
  
• Incubate 5 min at RT.
• Add 300 ul Solution III to neutralize.
• Mix really well by inverting >10 times or vortex gently at low speed.
  
• Incubate on ice for 10 minutes.
• Spin at top speed in the cold room for 30 min.
• Remove 800 ul supernatant to new tube.  Discard pellet.
  
• Add 700 ul cold isopropanol.
• Mix by vortex vigorously.
• Incubate on ice for 30 min.
• Spin at top speed in the cold room for 30 min to obtain a  tiny white DNA pellet (sometimes could spread on the side of tube).
• Discard supernatant.  Make sure to remove all supernatant to reduce residual protein carryover to the next step.
  
• Add 500 ul 70% ETOH to wash DNA pellet.
• Spin at top speed in the cold room for 15 min.
• Wash again by adding 500 ul 70% ETOH.  Make sure to remove all supernatant to reduce residual protein carryover to the next step.
• Spin at top speed in the cold room for 15 min.
• Remove ALL liquid (important to use tips to remove residual liquid to decrease contamination & improve quality of the miniprep DNA).
• Air dry.
• Add 100 ul 10mM Tris pH8.8.
• Incubate for 30 min at RT.
• Resuspend DNA by flicking tube & vortexing during the 30 min.
• Quick spin to collect all liquid to bottom of tube.
• Nanodrop.
• Label tubes : conc DNA, ligation of vector & insert used, date.
  
• Store miniprep DNA at -20 degrees if not used immediately.

• To check whether the PCR product has been successfully inserted into the vector, digest miniprep DNA (using the same enzyme sites for cloning).
• Make sure all buffers & miniprep DNAs are thawed completely (room temp is OK), then mix, vortex, quick spin to collect liquid at the botton of tube.  Then put everything on ice.
• For a 20 ul digestion reaction: use 2 ug DNA + 2 ul 10xNEB buffer + 0.2 ul 100xBSA + water.
• Calculate the volume of plasmid DNA needed from miniprep DNA concentration then calculate how much water is needed to make 20 ul. 
• Mix all ingredients well (by pipeting or vortex).
• Spin tube to remove bubbles.
• Add 1 ul of each enzyme per 20 ul digest reaction.
• Incubate at 37 degrees waterbath  for 1 hour.
  
• Run 1.2% Agarose TBE gel.  General guideline: Use 0.8% agarose for >2kb insert.  Use 1% agarose for 0.5-2 kb insert. Use 1.2% for <0.5 kb insert.
  
• If the gel shows 2 bands of expected sizes (the insert and the vector), we have a new "construct"!!
• Save all the minipreps that has the right insert.  Throw out the useless ones.
  

• Grow up the colony that has the right construct.
• Add 100 ul of the the bacterial culture from the original colony to 50 mL LB/antibiotics.
• Grow overnight at 37 degree shaking.
• Plasmid prep (scale up from miniprep accordingly; use 10 mL of solution I, II, III).
• Wash DNA pellet twice with 70% ETOH.
• Air dry.
• Resuspend DNA pellet in 500uL 10mM Tris, pH8.8.  Let soak for 1 hour.  Resuspend DNA pellet by pipetting up & down.
• Nanodrop.
• Label DNA/contruct name/vector+insert.
  
• Store DNA at -20 degrees.
• Final touch: digest 1 ug DNA/20 ul reaction to check insert.