File:Jr rmf 1.jpg

‘’’A’’’ 1.5% agarose gel with marker 100bp DNA ladder plus (Fermentas); pSB1A7: vector pSB1A7 digested with XbaI and SpeI according to [NEB] manual; RMF: PCR product of RMF amplified from DH5-alpha genomic DNA using forward primer 5'-cgcggaattcgcggccgcttctagatgaagagacaaaaacgagatcgcctgg and reverse primer 5'-cgcgctgcagcggccgctactagtattattaggccattactaccctgtccgc; reaction setup was 35ul water, 10ul [Finnzyme] Phusion HF buffer, 1ul 10mM dNTPs, 1ul 25uM forward primer, 1ul 25uM reverse primer, 0.5ul DH5-alpha genomic DNA, 0.5ul [Finnzyme] Phusion Hot-start polymerase; thermocycler program was 98C for 3min, cycle start: 98C for 10s, 55C for 30s, 72C for 10s, cycle end, 35 repeats, 72C for 30min, 4C hold; the PCR product was restriction digested with XbaI and SpeI and purified using Qiagen Qiaquick PCR purification kit; ligation: ligation of XbaI/SpeI linearized pSB1A7 with RMF insert. ‘’’B’’’ 2% agarose gel of test digests (XbaI/SpeI; according to [NEB] manual) of pSB1A7-RMF. Six colonies were used for overnight cultures and test digested; colonies were designated R1, R2, R3, R4, R5, R6. Only R2, R4, R5 and R6 contain the insert