Insertion molecular cloning in 24 hours, from design to purified DNA ready for transfection

Just cost 24 hours for the entire cloning process!

Here is how.

1. Design stage - 30 minutes: I have an design in mind and ready to realize it. Sit before my computer and open Vector NTI, which has all my DNA sequences information. My gene of interest is about 800 bp and I need it inserted into a retroviral vector. Luckily, the gene has been inserted into other vectors many times and I have several primers covering its 5' and 3' ends. Use spotlight in Mac OS X, I locate the primers' information - I store my primers as text files and desktop search engine, such as spotlight, has the full text index of it, which enable me to find THEM quickly.
2. Vector digestion setup - 10 minutes: I prefer to digest vector as completely as possible, even with those 5-minute or 15-minute Time-Saver. Recently, NEB has those enzymes branded as High Fidelity (-HF), in Buffer 4, which are really convenient for double digestion.
3. PCR reaction setup - 10 minutes: I used to use Novagen's KOD to amply inserts from either existing plasmids or cDNA libraries. Stratagene's Pfu Turbo is good for site-directed mutagenesis, but it is toooooo slow. KOD DNA Polymerase (formerly KOD HiFi DNA Polymerase) is a recombinant form of Thermococcus kodakaraensis KOD1 DNA polymerase, which is a high fidelity thermostable polymerase that amplifies target DNA up to 6 kbp with superior accuracy and yield! However, after I moved to UCSF, I start using a similar enzyme from Finnzymes, called Phusion High-Fidelity DNA Polymerase. The performance is awesome and the CCF here has a stock of it.
4. PCR reaction running while vector digestion in 37 degree - 1.5 hours
5. PCR product digestion while phosphatase treating the cut vector - 20 minutes: amplified insert doesn't require complete digested, 15 minutes is more than enough, especially for those Time-Saver. But, to avoid the single cut vector (maybe only 1% or 0.1% of all the digested DNA) show up as false positive on my plates, I prefer to do a phosphatase treatment for "short" cut vector. NEB's Antarctic Phosphatase is my choice, which can be heat inactivated.
6. Digested DNA running on agarose gel - 25 minutes
7. Gel extraction - 15 minutes
8. Ligation setup and quick ligation at room temperature - 10 minutes: usually I do 16 degree overnight ligation; but to save time, I use Quick Ligation Kit from NEB, which only needs 5 minutes at room temperature. Because there is PEG in the quick ligation reaction, it is very important to use high competent E.coli cells and avoid any heat inactivation after the ligation.
9. Heat-pulse based E.coli transformation - 30 minutes: I made my own competent cells every 6 or 9 months and store them in -80C freezer without any problem. I used to make electroporation competent cells following a chapter in Molecular Cloning. Recently I switched to make heat-pulse competent cells, which I think is more cost effective.
10. Transformed E.coli grown/selected on plate at 37 degree - 12 hours: My favorite E.coli strains are the SURE and SURE2 from Stratagene (now Agilent Technologies) - they grow fast and suppress unwanted recombination. One thing to keep in mind is that these E.coli are kanamycin resistant, nalidixic acid resistant and tetracycline resistant. For pEGFP-N1 vector series, I use DH5alpha.
11. Picked E.coli colony grown at 37 degree - 8 hours: SURE/SURE2 E.coli only needs 8-10 hours grow from single colony before getting enough bacteria for miniprep
12. Mini-prep DNA - 1 hour: Qiagen kits are expensive, which cost about $2 per prep. What I really need is some buffers, and DNA binding columns, such as UPrep Universal Spin Filters.
13. DNA digestion and running on agarose gel - 1 hour: I usually set up the digestion, and pour the agarose gel. When the gel is ready (about 20 minutes), the samples are ready as well.
14. Identify the correct clone and purified DNA is ready for transfection to mammalian cells

O, yeah!

Several notes

• I prefer overnight 16 degree ligation more than 5-min room temperate ligation. I usually only pick two colonies from a overnight ligated transformation and get the correct one. With quick ligation, I usually do 3 or 4, depending on how the plate looks like.
• To use the ccdB trick (such as making entry plasmids for the gateway system), special E.coli should be used, such as DB3.1 or ccdB Survival
• For some Dam/Dcm methylation sensitive enzymes, such as BclI or XbaI in certain condition, a dam-, dcm- strain of E.coli should be used, ie C2925 from NEB.
• Compatible cohesive ends I usually used: MfeI - EcoRI, SalI - XhoI, BglII - BamHI
• I am a big fan of NEB products. Although it is the only system I extensively used, it works. As long as it works, I won't change. People have suggest to me to use Gateway system, and I read about it and was excited about. However, using Gateway, I lose the happiness and joy from piercing DNA together one nucleotide by nucleotide - it is very important to me!
• When I design primers/oligos for cloning, I always ask myself the following questions: Do I need STOP code (TAA, which is part of HindIII)? Do I need ATG start code, or Kozak (GCCACC)? Are the fusion in frame with the original? Do I need a linker sequence, such as GGGS? Are the enzyme I selected presented in the vector, or in the insert? How specific is the oligo sequence? Do I need extra nucleotide at the end to help efficient digestion? ...

Feb 28, 2010 @ 23:00