Troubleshooting BioMagicTM Loading Buffer (Identify recombinants with our single buffer in 10 minutes!) Problem                  1. No plasmid visible on gel Note: high copy number plasmid may be replicated in low number due to intolerance of host cells. 2. Smear on gel   _____________________________ Troubleshooting Cloning No Colony Growth or Few/Weak Colonies If ligation is good: 1. inadequate time for growth 2. competency of cells and transformation procedure 3. selective antibiotics 4. cell death due to intolerance or plasmid recombination within cells 5. organic reagent contamination 6. PEG promotes ligation but inhibits transformation 7. saturation of competent cells 8. non-optimal volume transformed If ligation is suspected: 1. insert/vector are degraded prior to or during ligation 2. component of ligation is missing or bad 3. non-optimal insert & vector ratio 4. low-efficent ligation reaction 5. hidden restriction site(s)                   6. overdigestion 7. underdigestion If too many colonies without recombinant clone 1. antibiotic is bad.  AMP is not stable and only valid for 2 years as powder                        2. backgound caused by parental plasmids   If confirmation digestion yields fragment(s)not matching prediction 1. confirmation enzyme(s) being unreliable 2. plasmid rearragement due to high unstability within E.coli strain ______________________________ Trouble shooting PCR      If no PCR product 1. no template/too much template               2. annealing temperature 3. annealing time 4. template/primers 5. primers 6. cycles 7. gel sensitivity If smear product or non specific product    1. template 2. annealing temperature 3. annealing time 4. primers 5. cycles

How to Fix It Use 2-3ul ON culture or more instead of single colony. Colony may be junk due to invalid antibiotics. Vortex 10 seconds only with          BioMagic loading buffer ______________________________ grow additional 24-48hrs make sure + control plasmid works make sure to grow on the desired antibiotics lower growth of temperature lower antibiotic strength use BioMagicTM Gel-Free Cloning Kit   use BioMagicTM  Gel-Free Cloning kit remove PEG before transformation transform no more than 10ng DNA transform no more than 1/10 cell volume check DNA on gel try with + control ligation for sticky end fragment < 4kb, 3-5x for sticky end fragment > 4kb or blunt end fragment, 10-20x increase insert/vector ratio try our BioMagicTM Gel-Free Cloning Kit add PEG6000 or 8000 set up overnight ligation at 16C use high concentrated ligase sequence to rule out the possibility.  For example, desired cloning sites are A and B.  Hidden additional A could locate a few bases upstream of B or hidden B locating downstream of A without being detected on gel. It is more of an issue with non-commercial plasmids or ones being manipulated multiple rounds. 10-20 units (NEB) for 1ug up to 2-3 hrs. Longer digestion may cause problems such as star activity, chewed-back ends etc. use BioMagic Gel-Free Cloning Kit some enzymes such as NdeI are more sensitive than others to contaminants in DNA samples which inhibit enzyme activity test with competent cells alone use BioMagicTM Gel-Free Cloning Kit try different restriction site(s) for confirmation try BioMagic competent cells for free ______________________________ for plasmid, use 1ng-500ug.   for genomic, use 25ng-250ng (start with low)     lower annealing temperature increase annealing time check their sequence and make sure they anneal use IDT's primer analyzer to check the design (www.idtdna.com) increase PCR cycles (<35) load sufficient amount and soak in EtBr for 5-10 mins lower template Increase annealing temperature decrease annealing time increase primer specificity by increasing the length without compromising 2nd structure decrease PCR cycles