Subcloning 1. Overview 2. Method

Transcription

1 Subcloning 1. Overview: 1.1. Digest plasmids for vector + insert 1.2. CIP treat vector 1.3. Gel purify vector + insert 1.4. Ligate 1.5. Transform bacteria with ligation reactions 1.6. Qiagen or Eppendorf Miniprep 1.7. Qiagen Maxiprep 1.8. Solutions 2. Method: 2.1. Vector + insert digestion Digest the plasmids for the vector and the insert using the same restriction enzyme(s) or those that generate compatible ends As there are a couple of purification steps, through which you will lose some DNA, it is suggested that you begin by digesting 1µg of each plasmid CIP treat vector If digestion buffer and CIAP are incompatible, it may be necessary to gel purify or alcohol precipitate the DNA prior to CIP treatment To gel purify, pour a 1% agarose gel and run until your fragment is separated from the other bands and can be cut out of the gel Take a picture of the gel on the BioRad Gel Doc 1000 (Rm 7767) Excise fragments on the UV box (Rm 7779). Wear safety glasses Weigh a 1.5 ml microfuge tube for each sample Using a clean, sharp razor blade cut out the band representing your fragment. Trim away as much of the excess agarose as possible and transfer to microfuge tube Weigh the tube containing the agarose. Subtract the weight of the tube alone to obtain the weight of the agarose. DNA can be stored in gel, if necessary Gel Purify with QIAquick Gel Extraction Kit (Cat No ). Follow directions in manual and elute in 30-45uL dh 2 O Quantitate DNA using UV spec Alcohol precipitation is done by adding 2 volumes of ethanol and 0.1 volume of a high salt buffer such as 3M NaAcetate or TEN Mix thoroughly and for 30 minutes. Spin down at maximum speed in for 20 minutes Aspirate, wash with 500µL of 70% ethanol, and spin for 5 at max speed in microfuge Aspirate, dry pellet for 5-10 minutes at room temperature, and resuspend in an appropriate volume of ddh 2 O.

2 Add 1U CIP (Calf Intestinal Alkaline Phosphatase, Gibco BRL Cat. No ) to the vector digestion tube. If DNA was not gel purified or alcohol precipitated, it is not necessary to inactivate the restriction enzyme first Shrimp alkaline phosphatase (Roche Catalog # s and ) can also be used; it may prove to be a little more efficient than the calf intestinal AP Incubate 37ºC x 10min for vectors with a 5' overhang, or 50ºC x 10min for vectors that are either 5' recessed or blunt-ended Purify the vector + insert fragments Vector can be purified by gel purification, or phenol-chloroform extraction can be used alternately if the fragments were previously gel purified To gel purify, follow steps Ligation Set up ligation using ~30 fmol vector ends: ~120 fmol insert ends. To determine fmol, use the following equation: (ug DNA) x (10 6 pg / 1ug) x (1 pmol / 660 pg) x (1/ N) = (pmol DNA) N = number of bp 1 pmol DNA = 2 pmol ends 1 pmol (10-12 mol) = 1000 fmol (10-15 mol) Include vector only and no ligase controls Typical ligation reaction: 10.0 ul 2X Ligase Buffer? ul Insert (120 fmol; depends on concentration)? ul Vector (30 fmol; depends on concentration)? ul dh 2 0 (bring total volume to 20 ul) 0.5 ul DNA Ligase, HC (high concentration, 20 units/µl), or 1.0µL Ligase (5 units/µl) 20 ul total volume Incubate at room temperature for 1 hour Transformation of competent ElectroMAX DH5α-E cells with ligation reaction Follow Electroporation protocol Minipreps (while the protocol from Maniatis et al can be used, the kits are much quicker and provide a clean enough product for subcloning) Aliquot 5ml of LB + 10uL Amp (50mg/ml stock for final concentration of 100µg/mL) into microfuge tube Inoculate each tube with a single colony from the ligation reaction plates Grow O/N in the 37 o C 250RPM incubator.

3 Freeze down an aliquot of each miniprep (500ul bacteria + 500ul 2X Freezing solution) for growing up more plasmid at a later date. Store in the -80 o C Aliquot 1.5ml of each grow-up into a 1.5ml microfuge tube, and spin at max speed (~14,000 RPM) for 30 sec Follow instructions provided with Qiagen Miniprep Kit or Eppendorf FastPlasmid Kit and elute DNA in 30-50µL of ddh 2 O Set up restriction enzyme digests and run agarose gels to determine which of the clones contain the insert in the appropriate orientation and location Maxipreps Once you ve determined that a particular clone is correct, inoculate 250ml of LB + 500ul Amp (50mg/ml stock for final concentration of 100µg/mL) for a Maxiprep by adding ~500µL-1mL of the culture from Depending on the plasmid, you may need to grow up 2 x 250mL of bacteria for a good yield upon Maxiprep Grow O/N in the 37 o C 250RPM incubator and then follow the Qiagen Maxiprep protocol for purifying the DNA.

4 3. Solutions 3.1. LB Medium: 250 ml 500 ml 1L Bacto Tryptone 2.5 g 5.0 g 10.0 g Bacto Yeast Extract 1.25 g 2.5 g 5.0 g NaCl 2.5 g 5.0 g 10.0 g Bring to appropriate volume with dh 2 O and autoclave Allow medium to cool to about 60 o C before adding antibiotics and pouring plates; this is cool enough to hold the flask in your palms for ~2-5 seconds Add antibiotics (typically ampicillin, which should be added to a final concentration of 100µg/mL) LB Agar: LB medium (see above) + 1.5% agar: 250 ml 500 ml 1L 1.5% Agar 3.75 g 7.5 g 15 g Bring to appropriate volume with LB medium and autoclave Allow medium/agar to cool to about 60 o C before adding antibiotics and pouring plates; this is cool enough to hold the flask in your palms for ~2-5 seconds Add antibiotics (typically ampicillin, which should be added to a final concentration of 100µg/mL) Pour ~15-20mL medium into each petri dish Allow to cool, and o C upside-down to prevent moisture accumulation on top of the solidified agar Ampicillin: Stock Amp is 50 mg/ml, which is 500X for liquid medium and agar. In other words, add 2ul/ml of LB medium and LB agar Add ampicillin AFTER medium has been autoclaved and cooled to under 60 C.

5 3.4. SOB: Bacto Tryptone 10.0 g Bacto Yeast Extract 2.5 g NaCl g KCl g Adjust ph to 7.0. Bring to total volume 500 ml and autoclave Complete SOB includes MgCl 2 and MgSO 4, which are added AFTER the SOB has been autoclaved and cooled X MgCl 2 stock (1M): g MgCl 2 (hexahydrate) in 100 ml ddh 2 O. Autoclave X MgSO 4 stock (2M): g in 100 ml ddh 2 O. 0.22µm Filter SOC: Complete SOB media + 20mM Glucose X glucose stock (2M): g in 100 ml ddh 2 O. 0.22µm Filter.

6 3.9. 2X Freezing Medium MW (g/mol) 250 ml K 2 HPO g Sodium Citrate Dihydrate g MgSO 4 *7H 2 O g (NH 4 ) 2 SO g KH 2 PO g glycerol (ultrapure) g Aliquot into 100 ml bottles Cover with foil Sterilize by autoclaving Store at 4 o C.