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Making a cDNA construct from PCR product:

 General considerations:
  • 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.
Blue filter purification of PCR product:
  • Use recycled filters for PCR reaction.
  • Mix PCR reaction with QX1 well/ vortex/ quick spin.
  • Add 0.5 ml 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.

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.
Run DNA samples on 1.2%Agarose 1XTAE or 1%Agarose 1XTBE gel:
 
Blue filter purification of TAE gel slices:
  • 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.
Ligation:
  • 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 colonies for miniprep:
  • 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.
Digest miniprep DNA:
  • 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.
Making DNA stock:
  • 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.
YOU ARE FINALLY DONE WITH MAKING ONE CONSTRUCT!!!
 

Vivian Tang Lab Notebook 2013