SUBCLONING

INSERT

• Cut the amout of the plasmid containing about 1 ug of your insert (5 to 10 ug of total plasmid) with appropriate restriction enzymes in the total volume of 30 ul (single or double digestion) for 3 hours.
• Treat end if necessary as discribed below.
• Run low meltine point (LMP) agrose gel in 1X TAE. For fragments less than 200 bp, use 1.5% agrose gel; for 200-500 bp fragments, use 1.2%; for fragment over, use 1%.
• Cut the gel contain the fragment as small as possible.
• Extract the gel with gene clean method and elute DNA fragment in 15 ul of H2O.

VECTOR

• Cut 3 ug of vector plasmid with appropriate restriction enzymes in the total volume of 30 ul for 3 hours.
• Treat end including dephosphorylation if necessary as discribed below.
• Run 1% low meltine point (LMP) agrose gel in 1X TAE buffer. Run at 40 volts (gel may be melted at high voltages) till your vector run to over half the way through the gel.
• Cut the gel contain the fragment as small as possible.
• Extract the gel with gene clean method and elute DNA fragment in 30 ul of H2O.
• LIGATION

0.1 ug of vector fragment (Def = 1 ul)
excess amount of insert (Def = 7 u
1 ul of 10 X ligase buffer (need to be completly thawed)
1 ul ligase
mix in 0.5 ml tube and incubate at 16 C for over 8 hours.

TRANSFORMATION

• Take a 0.2 cm cuvette, lebbel on the top of cuvettes and cool it on ice.
• Get a tube (40 ul) of competent cells (Topp 10) from -70 C freezer and thaw the cells (do not over thaw them).
• Add 1 ul of ligation mix into the competent cells
• Mix well and transfer them into the cuvette (on ice).
• Set-up the Gene pulser at 25 uF, 200 ½and 2.5 kv.
• Wipe off the moisture outside the cuvette and push the slide into the chamber
• Push both red buttoms at the same time till your hear the sound.
• Immediatly add 1 ml of SOC into the cuvette and quickly resuspend the cells.
• Transfer the cells into a 15 ml round bottom tube and incubate them at 37 C for 1 hous.
• Plate 200 ul on LB plate containing 100 mg/ml Amp. If you want to plate all the cells, you can transfer 1 ml of the culture into a 1.5 ml microcentrifuge tube and spin for 30 seconds at 12 Krpm, take off extra medium, resuspend the pellet in 200 ul of medium and plate all of them.
  

END TREATMENTS

BLUNT a 5' overhand site

• Heat inactivate the digestion reaction at 65 C for 15 min.
• Spin for 5 seconds at 14 Krpm.
• Add 1 ul of klenow and 18 ul of H2O into 30 ul of digestion mix
• Incubate at 37 C for 5 min
• Add 1 ul of 10 X dNTP (2mM)
• Mix well.
• Incubate at 37 C for 20 min.
• Run LMP gel directly, or Pheno/Chlorform extraction and ETOH precipitation before further modification such as dephosphorylation.

BLUNT a 3' overhand site

• Heat inactivate the digestion reaction at 65 C for 15 min.
• Spin for 5 seconds at 14 Krpm.
• Add 3 ul of 10 X dNTP (2mM), 1 ul of T4 DNA polymerase and 16 ul of H2O into 30 ul of digestion mix.
• Mix well.
• Incubate at 37 C for 20 min.
• Run LMP gel directly, or Pheno/Chlorform extraction and ETOH precipitation before further modification such as dephosphorylation.

DEPHOSPHORYLATION

• 30 ul of digested vector after heat inactivation plus 14 ul of H2O; or resuspend ETOH precipitated DNA in 44 ul of H2O.
• Add 5 ul of 10X CIP buffer
• Add 1 ul CIP
• Incubate at 37 C for 30 min
• Run LMP gel directly.

DNA FRAGMENT PURIFICATION BY GENE CLEAN METHOD

• Put the gel slide (in LMP agrose with 1XTAE) containing your DNA fragment into a 1.5 ml microcentrifuge tube.
• Add 1 ml of NaI
• Heat at 65 C for 10 min.
• Add 8 to 10 ul of beads (resuspend completely before aliquoting)
• Vortex continousely for 5 to 15 min.
• Spin at 12 Krpm for 10 seconds
• Pour off liquid
• Resuspend the pellet in 1 ml of New Wash Buffer.
• Vortex for 5 min.
• Spin at 12 Krpm for 10 seconds
• Pour off liquid
• Resuspend the pellet in 1 ml of New Wash Buffer.
• Vortex for 1 min
• Spin at 12 Krpm for 10 seconds
• Pour off liquid
• Spin again at 12 Krpm for 10 seconds
• Use pipetman to suck off all the liquid.
• Resuspend the pellet in X amonut of H2O.
• Heat at 65 C for 10 min
• Spin at 12 Krpm for 10 seconds
• Transfer the supernatant (containing your DNA) into a new tube (0.5 ml).
• Spin at 12 Krpm for 10 seconds.
• Transfer the supernatant into a new tube (0.5 ml).

CLONING PCR PRODUCTS INTO EXPRESSION VECTORS

Oligo Design: XXX or XXXXXX (see p238 NEB Catalog) + inframed restriction sites
+ Specific sequences.
If the vector is not 5'end tagged, you need to put Kozak sequence to the
Amino terminal between the restriction site and the specific sequences
(ACCATG)

PCR: Use Pfu or Taq DNA polymerase.

Purification of PCR production:

• Transfer 1 to 5 tubes of PCR reactions into 0.5 or 1.5 microcentrifuge tube
• Add equal amount of chloroform and vortex.
• Spin at 14 Krpm for 3 min
• Transfer the TOP layer into a new tube.
• Add 1/10 Volume of 3M NaOAC
• Add equal amount of Isopropanol and Vortex for 1 min
• Spin at 14 Krpm for 15 min.
• Discard the Supernatnant
• Add 1 ml of 70% ETOH (carefully so you do not disrupt the pellet)
• Spin at 14 Krpm for 1 min
• Discard the Supernatnant (carefully so you do not thow away the pellet)
• Dry the pellet in the speed-Vac.
• Resuspend the DNA pellet in 40 to 43 ul of H2O.
• Add 5 ul of 10X restriction buffer
• Add 2 to 5 ul of restriction enzymes (do not go more than 5 ul total).
• Incubate at 37 C for overnight.
• Run LMP agrose gel (in 1XTAE).
• Gene clean and elute the DNA fragment into 15 ul of H2O.
• Use 7 ul for ligation.

GST fussion proteins containing peptides less than 20 amino acids

• Vector: 3 ug pGST2T cut with R1/BamH1(No dephosphorylation), purified from LMP Agrose gel, resuspend the DNA fragment in 30 ul of H2O.
• Insert: 16 ul of 1 ug/ul of Oligo1 = GATCC(XYZ)
  16 ul of 1 ug/ul of Oligo2 = AATT(Z'Y'X')G
  1 ul of 5 M NaCl
  Mix and put into 1L of boiling water and cool down slowly to RT (at least 3 hours)

Ligation: 1 ul vector
1 ul insert
1 ul 10 X buffer
6 ul H2O
1 ul T4 DNA ligase
16 C for > 8 hours.
Transformation: eletroporation as usual.

• Pick up about 10 colonies plus the GST2T control and grow in 2 ml of LB/Amp for Overnight.
• Inoculate 25 ul of O/N culture into 500 ul LB/Amp in a 5 ml tube.
• Incubate at 37 C for 2 hours
• Add 2 ul of 100 mM IPTG
• Incubate for another 2 hours
• Transfer 200 ul of the IPTG induced culture into a 1.5 ml tube.
• Spin at 14 Krpm for 2 min
• Resuspend the pellet in 40 ul of 1 X SDS loading buffer
• Boil the sample for 5 min
• Load on SDS-PAGE (12%) and run till the BCP dye to bottom.
• Stain with Coomassie Blue and Destain (you may Heat in microwave to get a quick answer).