Im doing research about gene fusion of 2 fragment ( 1000 bp and 500 bp ). I get single bright band from each gene fragment. I continue with 2 step PCR overlap PCR : first primerless PCR and the second PCR using forward and reverse primer.
i have calculated equimollar ratio for overlap reaction, but the product always show 3 fragment ( fused gene at 1500 bp, and 2 band of fragment residues ). So, I purify the 1500 bp band using gel extraction (qiagen) and get poor recovery (10-20 ng). I also purify the product of restriction enzyme digestion and get lower DNA concentration (5 ng/ul) as well as bad DNA Quality (worst 230/260 & 260/280). Would you please give suggestion what I need to check ?. Thank you in advance.
you may be getting low OD260/280 because some RE is still binding to the template dna. Adding a small amount of sds to release the RE before pruification might help.
You could increase the yield of the qiagen purification by adding hot (70c) elution buffer for 2x as long as the kit recommends to increase the amount of purified product. Also make sure you blank the specrometer with elution buffer not TE or water to ensure accurate determination of dna. A few ng of short (1500bp) is very many copies of the fragment and will be enough for many pcr reactions if that is your next step
Hello Rasyid, I am surprised that I have not heard of overlap PCR before (neat!). Alternatively, you can use RecA-independent recombination or RAIR cloning: https://www.jbc.org/article/S0021-9258(20)33772-8/pdf. RAIR cloning relies on homology-based recombination. The 5’ and 3’ ends of your amplicons need to be homologous with one another with melting temperatures (for the homologous regions) of 55 degrees Celsius. I have had great luck with RAIR cloning and it is relatively straightfroward. Following PCR of each fragment and vector, you just add the amplicons to heat-shock cells, perform hear shock protocol, plate the cells, and select positive clones via colony PCR.
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