Molecular Cloning Protocols

Digestion

Material

• EcoRI, BglII, PstI, XhoI, SpeI,XhoI,FD buffer,FD Green buffer, DNA,sterile water

Digestion temperature

• 37℃

Time period

1. Plasmid: 4-6h(the higher the concentration, the longer time it takes.
2. DNA fragment:4-6h(the higher the concentration, the longer time it takes.Since it is more difficult to digest DNA than plasmid, digesting DNA requires more time.
3. Hind III, EcoR I, BamH I: 0.5-1 hour. No longer than 1 hour.

System amount

1. 50ul system is preferred if there is subsequent operations. If digestion is only for identification, 20ul system is used.
2. During the double digestion, the optimum buffer system which is suitable for both enzymes should be utilized. Without such shared system, digestion of two different enzymes is usually separated.

System

Enzyme	1μl
10×FD Buffer or FD Green buffer	5μl
DNA	≤2μg
Sterile water	up to 50μl

Gel extraction

Material

• Agarose Gel DNA Extraction Kit (centrifugation)

Experimental Procedure

1. Add 500uL balance liquid(BL) to the spin column(CA2). Centrifuge at 12,000 x g for 1 min at room temperature. Discard the flow-through and put the column back to the collection tube.(optional)
2. Excise the DNA fragment from the agarose gel and weigh it in a clean centrifuge tube (A gel slice of 100 mg approximately equals to 100 µL).
3. Add 3 volume of sol solution(PN) to the centrifuge tube and incubate the mixture at 50℃ for 10 min with mixing the tube by tapping the bottom of the tube occationally till the gel has melted completely. Cool the tube to RT.
4. Transfer the mixture to an spin column (CA2) within a collection tube. Centrifuge at 12,000 x g for 1 min at room temperature. Discard the flow-through and put the column back to the collection tube.
5. Add 600 µL DNA Wash Buffer (PW) to the column and centrifuge at 12,000 x g for 1 min at room temperature. Discard the flow through and put the column back to the collection tube.
6. Repeat step 5.
7. Put the column back to the collection tube. Centrifuge at 12,000 x g for 2 min at room temperature to discard the PW totally. Keep the lid open for several minutes.
8. Put the column back to the clean collection tube. Add ddH2O to the column vacantly and keep it at room temperature for 2 minutes. Centrifuge at 12,000 x g for 2 min at room temperature to collect the DNA solution.
9. Transfer the eluted solution to CA2 and keep it at room temperature for 2minutes. Centrifuge at 12,000 x g for 2 min at room temperature.
10. Gel electrophoresis to verify the correctness and concentration of bands.

Important Notes

1. If the balance liquid turns turbid, incubate it at 37℃ for several minutes.
2. When cutting the gel, keep the UV irradiation time period as short as possible, otherwise DNA would be damaged.
3. For those DNA fragment shorter than 100bp or larger than 10kb, the sol solution volume should be increased as well as the time period for adsorption and elution.
4. Recovery depends on the initial DNA amount and elution volume. The less the DNA, the less the elution volume, the smaller of recovery.

PCR

Reagents

• KOD-Plus-Neo
• Tap enzymes

Experiment system

• Experiment system with KOD enzymes (Mostly 50uL)

• Experiment system with taq enzymes:

• Reaction temperature:

        Two-step method:
        Predenature
          94℃，2min
        Denature and extension
          {
          98℃ 10sec 
          68℃ 30sec/kb
          }
                        × 25-45 cycles
          4℃

        Three-step method：
        Predenature
          94℃，2min
        Denature，annealing and extension
          98℃ 10sec   
         {
         （Tm-5）℃  30sec
          68℃  30sec/kb
          }
                       × 25-45 cycles
          4℃

Important Notes

1. KOD enzymes should be operated in ice.
2. Two-step method is preferred if primer Tm is relatively high.
3. 1.2% agarose gel is used to separate 1kb-5kb DNA; 0.8% agarose gel is used to separate 3k-10k DNA; 2.5-3% agarose gel is used to separate DNA longer than 100bp.
4. PCR reaction system varies from 20uL to 100uL. Under most cases, 20uL system is preferred as long as PCR is for identification only. However, when subsequent experiments such as tailing, digestion which require certain amount of DNA are necessary, 50uL system is usually preferred.
5. KOD enzymes are preferred if the purpose of PCR is for gene amplification. At the same time, PCR for identification or tailing usually requires taq enzymes.

PCR Purification

Reagents

• LifeFeng DNA PCR purification kit

Experimental Procedure

1. Transfer PCR system (< 250uL) to 1.5mL microcentrifuge tube. Add 5 volume Buffer PD and mix. Have a quick centrifugation to remove the liquid on lid.
2. Transfer the mixture to DNA spin column (A4) and put it in a collection tube.
3. Centrifuge at 7000g for 1 minute and replace the collection tube with a new collection tube.
4. To wash, add 500uL Buffer WB to the spin column (A4) and centrifuge at 7000g for 1 minute. Discard the flow-through and place the column back in the same tube.
5. Repeat step 4.
6. Centrifuge at 12,000g for 2 minutes.
7. Place spin column in a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add ddwater (>30uL) to the center of the membrane. Let the column stand for 1-2 min and centrifuge the column for 1 min at 12,000g.
9. Add elution liquid back to spin column and centrifuge at 12,000g for 1 min. ( Inoculating ddwater at 65℃ prior would improve the elution efficiency )
10. Gel electrophoresis to verify the concentration of bands.

Important Notes

• Buffer PD contains irritant compounds, thus be careful not to avoid contamination of skin.

DNA Ligation

Experimental Procedures

1. Set up the following reaction in a microcentrifuge tube on ice.
2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours
5. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

Ligation System

COMPONENT	20 μl REACTION
10X T4 DNA Ligase Buffer	2 μl
Vector DNA	0.025 pmol
Insert DNA	0.076 pmol
T4 DNA Ligase	1μl
Nuclease-free water	to 20 μl

Bacteria Culture

Strain

1. DH5a （F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–）use for cloning
2. BL21(DE3) (fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS,λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5) use for expressing

Medium

LB Medium

NaCl	10g
Tryptone	10g
Yeast Extract	5 g
Water	up to 1000mL

SOB Medium

Tryptone	20g
Yeast extract	5g
NaCl	0.5g
1M KCl	2.5ml
ddH2O	up to 1000 ml

Adjust pH to 7.0 with 10N NaOH, autoclave to sterilize, and add 10 ml of 1 M MgCl2 before use

Antibiotics

	Storage	Use
Kana	50 mg/ml(Water)	50 μg/ml
Amp	50 mg/ml(Water)	50 μg/ml
Tet	10 mg/ml (Alcohol)	10 μg/ml
Cm	34 mg/ml (Alcohol)	68 μg/ml

Transformation

1. Remove competent cells from freezer and allow to thaw
2. Add 3-5 μl of DNA to the cells
3. Incubate on ice for 30 min
4. Heat shock the cells at 37°C for 1.5 mins
5. Add 500 μl of SOB medium, and incubate in a shaker at 37°C for 1 hour
6. Centrifuge for 3 min at 3,000 g, remove and keep 100 μl of the supernatant, and pour away the rest
7. Re-suspend the pellet in the 100 μl of supernatant that was removed earlier
8. Spread All onto one plate with corresponding antibotics, and then pour the rest onto another. Incubate the plates overnight at 37°C.