LIC Cloning (Ligation independent cloning)

Procedure

Design primers compatible for cloning in vector containing the LIC sequence (e.g. pDNR Dual LIC) and perform PCR. Separate 4 µl of PCR reaction via agarose gel electrophoresis to check PCR product. Many unspecific bands besides the desired one require gel separation of the PCR sample and extraction of the desired band. If necessary, 2-3 PCR reactions can be pooled to gain more material. Add 10x NEB2 Buffer to the PCR sample to reach 1x end concentration and add 2 µl of DpnI enzyme. Incubate 4 h at 37°C and inactivate enzyme at 80°C for 10 min. Purify PCR product with Qiagen PCR purification Kit. Determine concentration of PCR product at Nanodrop and calculate molarity.

Molarity Calculation for LIC Cloning

The molar concentration of the PCR product is calculated as follows:

$c_{mol}=\frac{c_{PCR}\times 10^9}{N_{bp}\times 650}$

(Equation 1)

Variables:

• $c_{mol}$ = molar concentration [pmol/μL]
• $c_{PCR}$ = PCR product concentration [ng/μL]
• $N_{bp}$ = PCR product length [bp]
• $650$ = average molecular weight per base pair [g/mol]

Example: For a 1500 bp PCR product at 50 ng/μL:

$c_{mol}=\frac{50\times 10^9}{1500\times 650}=51,282\text{ pmol/μL}$

T4 Treatment

✓ Reaction mix

☐	PCR product	~0.3 pmol
☐	10X NEB buffer	4 µL
☐	dCTP (100 mM)	1 µL
☐	DTT (100 mM)	2 µL
☐	ddH2O	depends
☐	T4 Polymerase	1 µL
☐	Total	up to 40 µL

• Incubate for 30 min at 22°C and 20 min at 75°C (PCR machine recommended)
• Incubate PCR product with T4 polymerase-treated vector (aliquots frozen at -20°C)
  • Pre-treated vector – 2 µl (~50 ng)
  • PCR product – 8 µl
• Set up a vector-only control w/o PCR product as well as a PCR product only control w/o vector
• Incubate 10 min at room temperature, add 1 µl 100 mM EDTA and incubate another 10 min.
• Transform E. coli NovaBlue with complete reaction mix.