Growing Up Plasmids
A) Transformation of E. Coli
-Chemically competent DH5α aliquots (50 μL)
-LB Broth (or SOC)
-LB agar plate containing appropriate antibiotic
Thaw appropriate number of chemically competent DH5α by placing on ice
Add 1-5 μL plasmid (depending on concentration) to thawed cells and mix gently.
Place on ice for 5-30 minutes.
Heat shock cells by placing in 42 ̊C bath for 30 seconds or 37 ̊C for 2 minutes.
Place immediately on ice for 2 minutes (Place SOC in 37 ̊C water bath to warm up).
Add 1 mL room temperature LB Broth (or 300 μl23o00- SOC) to cells and place in 37 ̊C shaker for an hour. Place at an angle to increase surface area.
Plate out 100-200 μL of culture on plate containing appropriate antibiotic and incubate overnight at 37 ̊C upside down, with agar on top (to prevent accumulation of condensation on the agar). Store remaining bacteria in 4 ̊C cold room in case mote is needed to replate.
Next morning check for colonies. Wrap edges of plate with parafilm to prevent desiccation and place in 4 ̊C cold room until needed. If there are very few colonies (or none), one can concentrate the remaining transformed bacteria by spinning 1 min/1000 rpm, resuspending pellet in 100 μL and plating entire volume.
B) Preparing mini culture
-LB plate containing distinct colonies
-LB broth containing proper antibiotic (Ampicillin stock is 1000x, Kanamycin is 500x)
-Filtered 20 μL/200 μL tips or inoculation loop
-14 mL round bottom tubes
1) Prepare mini culture the day before and let it grow overnight
Aliquot 3-5 ml of LB broth with antibiotic into round bottom tubes
Carefully pick a bacteria colony using either the inoculation loop (touch the colony) or the tip by stabbing the agar (do not touch more than one colony)
Shake inoculation loop inside the LB broth to detach the colony from the loop. If a tip is being used release it into LB broth. Replace cap on tube but leave it loose.
Place tubes in 37 ̊C shaker overnight.
Next day, remove tubes from shaker, tighten caps and place at 4 ̊C until needed.
2) Prepare mini culture in the morning and removing 8 hours later checking for cloudiness in the broth with same techniques as listed above.
C) Preparing Maxi culture
-LB broth containing antibiotic (Ampicillin stock is 1000x, Kanamycin is 500x)
Add proper antibiotics to 200 mL of LB broth in a flask (200 μL Amp)
Add 1 mL of day mini culture or 500 μl of overnight mini culture to flask
Place culture in 37 ̊C overnight
After 14-16 hours of growth, store at 4 ̊C or use immediately.
D) Maxi prep of DNA
-Qiagen Maxi prep kit
-70% ethanol (made by diluting 100% ethanol)
Follow directions in the kit except:
Following elution add 1.5 mL sodium acetate and fill tube to 50 mL with 100% ethanol. Place overnight in -20 ̊C freezer. Next day spin down at 4 ̊C at maximum centrifuge speed. Wash by removing supernatant and adding 5 mL 70% ethanol to the pellet. Vortex to remove pellet from the bottom of the tube, then spin for 5 minutes. Remove supernatant and dry upside down for 10 minutes. Carefully resuspend DNA in 500 μL TE.
E) Quantification if DNA using UV Spec
-Plasmid to be quantified diluted 1:50 in TE (2 μL plasmid: 98 μL TE)
-Two quartz cuvettes
Rinse out cuvettes with ddH2O and dry by gently taping on kim-wipe as well as carefully removing remaining liquid with a micropipette.
Add 100 μL 1x TE to chipped cuvette to serve as a blank, place blank in first slot of spec
Add diluted plasmid to other cuvette, place cuvette in the second slot of spec
Using program 4 on spec, measure absorbance at 260 and 280.
260/280 should be around 1.7-2
Take absorbance at 260 and multiply by 1.25 to get the concentration (μg/μL). If diluted 1:100, multiply by 2.5. If diluted 1.35 multiply by 0.75 etc.
F) Verify identity by restriction enzyme analysis
-Plasmid(s) to be analyzed
-Appropriate enzymes (always kept on ice)
-10 X buffer and 10x BSA (if needed)
Determine the appropriate enzymes to best verify the identity of your plasmids.
If using more than one enzyme in a digestion, check NEB chart to identify the best buffer to use for the reaction. Also, if one enzyme requires 10x BSA, you must use it in the reaction even if the other doesn’t (BSA won’t affect the activity of the other enzyme.
If performing the same digestion on more than one plasmid, make up a master mix as in the following example. In this example there are 10 plasmids to test and the digest is 1 μL of each with BamHI and HindIII together.
Once the master mix is made, aliquot appropriate amount of mix into a clean 1.5 mL tube. In the previous example, the added amount of the mix would be 19 μL. Then add the appropriate amount of plasmid. In the previous example, the added amount of the plasmid would be 1μL. Place reactions in a 37 ̊C water bath for 1 hour.
Pour 0.7% agarose gel with the appropriate amount of lanes. Let it cool until solid (about 30 minutes). Add 6x DNA LB (in the previous example the amount would be 4 μL) to samples and load the gel along with a ladder. When ran take a picture of the gel.
If identity of plasmid is verified, label and place in appropriate plasmid box slot.