Cloning Protocol

Cloning Protocol Ian Hewson 2002 (modified from protocol in Promega P-gemT Easy Vector Cloning kit)

Preparation for Cloning: Ampicillin 1g ampicillin 10 ml deionised water Add amp to DIW, filter sterilize through 0.2 um syringe filter. Aliquot into 1 mL aliquots and store at – 20 deg C until use LB media 10g Tryptone 5g Yeast Extract 5g NaCl 1000ml deionised water Make 2 x 500 mL volumes for every 2 samples to clone. Autoclave then cool to 50 deg. C. Add 1 mL 100 mg /ml ampicillin to make 100 ug / ml LB / amp. To one of the 500 ml volumes add 15 g agar , when cool add ampicillin then pour 25 ml into petri dishes (75 mm variety). NOTE: if you want to use S-Gal instead of X-Gal then use the entire contents of one of the commercially available S-Gal mixes, Just remember to add ampicillin when cool. You should have ~ 4 plates per sample to be cloned. SOC media 2g tryptone 0.5g yeast extract 1ml 1M NaCl (make first) 0.25ml 1M KCl (make first) 97mL deionised water Dissolve then autoclave. On its own, stable indefinitely at 4 deg C. Day of use, make up 2M Glucose (50 mL) and 50mL 2M Mg solution (10.15g MgCl2-6H20 + 12.35g MgSO4-7H20 + 25 mL DIW). Filter each of the glucose solution and Mg solution. Add 1mL of each of glucose and Mg solutions to the SOC media, then filter sterilize (0.22 um) the entire media. Store at 4 deg C until 1h before use.

IPTG 0.12 g isopropyl-b-d-thiogalactopyranoside (IPTG) 5 mL deionised water Dissolve then filter sterilize through 0.22 um. store at 4 deg C. stable for many months

X-Gal 0.10g 5-bromo-4-chloro-3-indoly-b-d-galactoside (X-gal) 2 mL N,N-dimethylformamide Dissolve, cover in foil and store (stable for many months; do not filter) LAIX plates (LB/Ampicillin/IPTG/X-Gal) Take LB / amp plates into laminar flow, spread 100 ul IPTG onto plates and let adsorb by placing inverted in 37 deg C incubator for 30 min. After this, spread 20 ul of X-Gal onto plates, then let adsorb again at 37 deg C. Store plates at 4 deg C. Good for 1 week. Toothpicks Cut toothpicks into 2 pieces, put in beaker and autoclave with alfol over the top. Growth blocks Cover 96 – well polypropylene growth blocks in alfoil, then autoclave Also make sure you have ~ 100 eppendorf tubes (0.5 mL + 1.7 mL), and enough 5 mL round bottomed falcon tubes with which to work. We also use 1.7 ml Eppendorf Tubes if colony number < 96.

Cloning Protocol (adapted from p-GemT Easy Vector (Promega) kit) Preparation of Amplimer Dilute your target DNA to 2.5 ng/ul in DIW. Make the master mix: 10 X PCR Buffer MgCl2 25mM dNTPs (nucleotide mix) Primer 58 Primer 20 BSA (40 ng/ul) DIW Taq (Promega) Sample

10 ul 10 ul 2.5 ul 2 ul 2 ul 1 ul 67.5 ul 1 ul 4 ul

With unamended negative control.

Use the following cycling conditions: No hot start (you are band-isolating later) Denature 94 deg C for 30s Anneal 56 deg C for 30 s Extend 72 deg C for 90 s with a final extension step of 72 deg C for 4 min 4 deg C chill at end of cycle Run 25 cycles After PCR is finished, run a deep-well gel (done by using 60 mL of 0.5 X TBE + 1.5 % NuSieve agarose and using one of the small gel castings taped around the gaps – this stuff is really viscous). After gel has set (may take a while since gel temp is very low), put I gel box with 0.5 X TBE. Load the entire contents of the PCR reaction into one well + 10 ml loading dye mixed in the PCR tube. Run gel at 100 V for 1 h. Stain gel for 30 min with 20 ul / ml SYBR Gold. Take stained gel to large transilluminator (one with plastic shield). Prepare one 2 mL tubes for each sample. Wearing UV-resistant face mask and gloves, turn on transilluminator and off lights. Acting quickly (shortwave radiation causes formation of primer-dimers), excise each band and place in a 2ml tube. These can be stored for later use at –20 deg C, but in my experience it is better to get all the way through to ligation on day one. Extract excised bands using QIAGEN MinElute gel extraction kit (see kit protocol). Quantify DNA after elution using PICO green kit (see separate protocol). If low or no DNA, do not proceed. Calculate how much DNA you will need downstream using the following equation: 50 ng insert x insert size in kb 3.0 kb vector

x 3/1 = amt required

If ~ 10 ng/ul or more of product is obtained, proceed to next step. I have ligated using a ratio as low as 1/8 (successfully). Ligation Set up one ligation reaction per sample to be cloned, plus one background, and one positive control.

Before starting, if the Easy Cloning Vector II kit is new, aliquot the supplied ligation buffer into 100 ul aliquots and store at –80 deg C. This is really sensitive to freeze-thaw. Set up the following ligation reactions: Sample 2X rapid ligation buffer 5 ul pGem-T Easy Cloning Vector (50ng) 1 ul PCR Product 3ul max Control insert DNA T4 ligase 1 ul DIW -

Positive 5 ul 1 ul 2 ul 1 ul 1 ul

Background 5ul 1 ul 1ul 3 ul

Mix reactions by mixing with pipette tip – BE GENTLE. Use 0.5 mL autoclaved tubes for ligations! Put in refrigerator (4 deg C) overnight. Transformation Begin starting transformation, pre-heat 37 deg C shaker and also heat a water bath to 42 deg C EXACTLY!! You also need ice from the maker on Level 3. 1. Remove ligations from refrigerator and equilibriate to room temp (1 min). Centrifuge if necessary to the bottom of the tube. 2. Add 2ul of each ligation to the bottom of a sterile 5 ml falcon round- bottomed tube which has been pre-cooled on ice. Make sure tubes are labeled 3. Remove JM109 competent cells from freezer and place in a 50 % ice / 50 % DIW bath for EXACTLY 5 minutes. Mix by flicking GENTLY. Talk nicely to cells! 4. Add 50 ul competent cells to the falcon round- bottomed tubes on ice using widebore pipette tips and pipetting gently. flick gently to mix. 5. Leave on ice for 20 minutes. 6. Heatshock cells for EXACTLY 45 s at 42 deg C. Time is very very important! 7. Return to ice for 2 minutes EXACTLY 8. Add 950 ul SOC media (see first part of this protocol) to each transformation and mix by flicking GENTLY. Close tubes completely (airtight). Put in incubatorshaker (37 deg C) for 90 minutes.

9. After 60 minutes, put LAIX plates in 37 deg C incubator oven (i.e. not shaker) inverted with lids off to dry out for 30 mins. 10. After the plates are dry and the transformations have incubated for 90 minutes in the shaker, take to laminar flow hood. Using sterile pipette tips, spread 150 ul of the transformation cultures onto the LAIX plates. I do about 4 plates per sample (excess colonies are obtained, but plates can be kept at 4 deg C for later picking and growing). 11. Seal plates with parafilm and place in 37 deg C oven overnight.\

Colony screening and liquid culture 12. After plates have incubated, place in 4 deg C fridge for 30 min (allowes easier screening of blue / white colonies. 13. Prepare a growth block (96 well) by pipetting 1.2 mL of LB + Amp liquid media into each well using the 1250 ul multipipettor . You can use a reagent reservoir for this since you will have excess LB + Amp. Prepare as many blocks as you have samples. 14. Using sterile toothpicks, pick up WHITE colonies (not blue or light blue) and drop toothpick into growth block. Do this for as many colonies as you can find on the plate. 15. When all wells are full, seal with 96 well- sealing tape , making sure all wells are covered and sealed around the edges. Using a sterile 32 G needle, prick two small holes on the top of each well. 16. Place growth blocks into the 37 deg C incubator – shaker and incubate overnight. See “Cheap protocol for plasmid preps from clones” for next steps in preparing plasmids, and “ checking for inserts” for how to check for inserts.