Phenol Chloroform Extraction for Plasmid Isolation

• Initial protocol same as Plasmid DNA isolation.
• After the wash with 75% ethanol, suspend the pellet in 500ul of TE buffer.
• Add equal amount of phenol:chloroform:isoamyl-alcohol (25:24:1), and centrifuge at 10,000rpm for 10mins.
• Three layers will be observed, carefully take the upper aqueous layer without disturbing the middle white layer.
• Transfer that to another eppendroff and add equal volume of phenol:chloroform:isoamyl-alcohol (25:24:1), and centrifuge at 10,000rpm for 10mins.
• Again take the aqueous layer into another eppendroff and add equal amount of chloroform, centrifuge at 10,000rpm for 10mins.
• Remove the upper aqueous layer and add 1/30th volume of sodium acetate (pH5.2) and 0.7-0.8 volume of isopropanol.
• Centrifuge at 14,000 rmp for 30mins at 4 degree C.
• Carefully decant the supernatant, to the pellet add 100-200ul of 70% ethanol.
• Centrifuge at 14000 rpm for 10 mins, discard the supernatant and air dry the pellet.
• Add 50ul of TE buffer pH 8.

Questions & Answers

Check out the following link for some basic queries regarding AGE
Agarose Gel Electrophoresis

Plasmids

pUC18

pUC18 and pUC19 vectors are small, high copy number, E.coli plasmids, 2686 bp in length. They are identical except that they contain multiple cloning sites (MCS) arranged in opposite orientations. pUC18/19 plasmids contain: (1) the pMB1 replicon rep responsible for the replication of plasmid (source – plasmid pBR322). The high copy number of pUC plasmids is a result of the lack of the rop gene and a single point mutation in rep of pMB1; (2) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – plasmid pBR322). It differs from that of pBR322 by two point mutations; (3) region of E.coli operon lac containing CAP protein binding site, promoter Plac, lac repressor binding site and 5’-terminal part of the lacZ gene encoding the N-terminal fragment of beta-galactosidase (source – M13mp18/19). This fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic complementation with a defective form of beta-galactosidase encoded by host (mutation lacZDM15). In the presence of IPTG, bacteria synthesise both fragments of the enzyme and form blue colonies on media with X-gal. Insertion of DNA into the MCS located within the lacZ gene (codons 6-7 of lacZ are replaced by MCS) inactivates the N-terminal fragment of beta-galactosidase and abolishes alfa-complementation. Bacteria carrying recombinant plasmids therefore give rise to white colonies.

pCMV-b-gal

This is a high copy number eukaryotic vector, pCMVb expresses the full-length b-galactosidase gene under the control of the cytomegalovirus immediate early gene (CMV IE) promoter.  This vector is very useful for transfection of mammalian cells in culture and for use in other species.  The b-galactosidase enzyme expression is enhanced by elements including: SD/SA-RNA splice donor and acceptor sequence, and SV40 late polyadenylylation signal.  pCMVb expression vector also contains b-lactamase gene, which acts  as a selection marker (100mg/mL ampicillin resistance) in E. coli host.   pCMVb vector has been tested to generate up to 2530u/mg cell extract (MacGregor, and Caskey).  In addition, the b-galactosidase gene can be excised using the NotI sites to allow the insertion of other genes to be expressed under the same regulatory elements in mammalian cells.

pIRES2-EGFP

pIRES2-EGFP contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between the MCS and the enhanced green fluorescent protein (EGFP) coding region. This permits both the gene of interest (cloned into the MCS) and the EGFP gene to be translated from a single bicistronic mRNA. pIRES2-EGFP is designed for the efficient selection (by flow cytometry or other methods) of transiently transfected mammalian cells expressing EGFP and the protein of interest. This vector can also be used to express EGFP alone or to obtain stably transfected cell lines without time-consuming drug and clonal selection. EGFP is a red-shifted variant of wild-type GFP (3–5) which has been optimized for brighter fluorescence and higher expression in mammalian cells. The MCS in pIRES2-EGFP is between the immediate early promoter of cytomegalovirus (PCMV IE) and the IRES sequence. SV40 polyadenylation signals downstream of the EGFP gene direct proper processing of the 3' end of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pIRES2-EGFP backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
pIRES2-EGFP replaces (but is not derived from) the pIRES-EGFP Vector previously sold by BD Biosciences Clontech. pIRES2-EGFP is functionally similarly to pIRES-EGFP; however, pIRES2- EGFP gives brighter EGFP fluorescence than the older vector.

Transformation

Introduction

Transformation is a technique to introduce DNA into bacterial cells. There are many variations on a common theme, but the key points are listed below. Check details with supplier of competent bacteria and note that variations in timings and volumes will vary with application and bacterial strain.

There are four stages:

• Mix DNA/bacteria and incubate on ice Ð do not use an excessive amount of DNA, both in terms of concentration and actual volume (less than 1 μg and less than 10 μl). Note,protein (e.g. Ligase) will reduce transformation efficiency, but it is not always necessary to remove prior to transformation.
• Heat shock - necessary for DNA uptake. Time heat shock carefully - excessive heat shock will kill the bacteria and the transformation will fail.
• Recovery - prior to selecting for transformed bacteria with antibiotics, it is necessary to allow them to recover in rich medium (e.g. LB, SOC or 2YT) for 30-60 mins at 37 ûC.
• Selection - essential to isolate (as single colonies) the bacteria which have taken up DNA.This is usually performed on solid medium (LB-agar) in the presence of antibiotics. Cells are incubated at 37 ûC overnight.

Competent bacteria are extremely fragile - always thaw slowly on ice and do not hold the base of the eppendorf tube. The compency of the bacteria is also important - "sub-cloning efficiency" means about 106 colonies are produced per μg of (purified) DNA. "Library efficiency" can mean in excess of 109 colonies produced per μg of (purified) DNA. For sub- cloning and mutagenesis is normally sufficient, although "Library efficiency" bacteria may be useful if problems arise.

Bacterial Transformation

1. Add 1-10 μl of the DNA (Experimental reaction or positive/negative control) to a vial (20-200 μl) of competent E. coli cells and mix gently. Do not mix by pipetting up and down.

2. Incubate on ice for 30 min.

3. Heat shock the cells for 30 sec at 42OC without shaking (time varies by strain).

4. Immediately transfer the tubes to ice and incubate for 2 min.

5. Add 50-500 μl nutrient broth (room temperature).

6. Cap the tube tightly and shake the tubes at 37ûC for 30 min. Place on ice.

7. Spread 50-500 μl from each transformation on a L-broth agar plates containing antibiotics at the appropriate concentration. Incubate plates for 5-10 mins at room temperature, then invert the plates and incubate overnight at 37OC.

Most strains require 12-18 hours to form colonies. Do not incubate for excessive times as satelite colonies will form. Plates/colonies can be stored for a few days at 4 OC if not to be used immediately.

Calculate transformation efficiency for the 1X and 10X DNA concentrations using the formula below.

Miniprep Plasmid DNA Isolation

Plasmid DNA Isolation

Isolation of plasmid DNA from E. coli is a common routine in research laboratories. You will perform a widely-practiced procedure that involves alkaline lysis of cells. This protocol, often referred to as a plasmid "mini-prep," yields fairly clean DNA quickly and easily.

Procedure

1. Fill a microcentrifuge tube with saturated bacterial culture grown in LB broth + antibiotic. Spin tube in microcentrifuge for 1 minute, and make sure tubes are balanced in microcentrifuge. Dump supernatant and drain tube briefly on paper towel.
2. Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel.
3. Add 0.2 ml ice-cold Solution 1 to cell pellet and resuspend cells as much as possible using disposable transfer pipet.
   
   • Solution 1 contains glucose, Tris, and EDTA. Glucose is added to increase the osmotic pressure outside the cells. Tris is a buffering agent used to maintain a constant pH ( = 8.0). EDTA protects the DNA from degradative enzymes (called DNAses); EDTA binds divalent cations that are necessary for DNAse activity.
4. Add 0.4 ml Solution 2, cap tubes and invert five times gently. Let tubes sit at room temperature for 5 minutes.
   
   • Solution 2 contains NaOH and SDS (a detergent). The alkaline mixtures ruptures the cells, and the detergent breaks apart the lipid membrane and solubilizes cellular proteins. NaOH also denatures the DNA into single strands.
5. Add 0.3 ml ice-cold Solution 3, cap tubes and invert five times gently. Incubate tubes on ice for 10 minutes.
   • Solution 3 contains a mixture of acetic acid and potassium acetate. The acetic acid neutralizes the pH, allowing the DNA strands to renature. The potassium acetate also precipitates the SDS from solution, along with the cellular debris. The E. coli chromosomal DNA, a partially renatured tangle at this step, is also trapped in the precipitate. The plasmid DNA remains in solution.
6. Centrifuge tubes for 5 minutes. Transfer supernatant to fresh microcentrifuge tube using clean disposable transfer pipet. Try to avoid taking any white precipitate during the transfer. It is okay to leave a little supernatant behind to avoid accidentally taking the precipitate.
   
   • This fractionation step separates the plasmid DNA from the cellular debris and chromosomal DNA in the pellet.
7. Fill remainder of centrifuge tube with isopropanol. Let tube sit at room temperature for 2 minutes.
   
   • Isopropanol effectively precipitates nucleic acids, but is much less effective with proteins. A quick precipitation can therefore purify DNA from protein contaminants.
8. Centrifuge tubes for 5 minutes. A milky pellet should be at the bottom of the tube. Pour off supernatant without dumping out the pellet. Drain tube on paper towel.
   
   • This fractionation step further purifies the plasmid DNA from contaminants. This is also a good place to stop if class time is running out. Cap tubes and store in freezer until next class period.
9. Add 1 ml of ice-cold 70% ethanol. Cap tube and mix by inverting several times. Spin tubes for 1 minute. Pour off supernatant (be careful not to dump out pellet) and drain tube on paper towel.
   
   • Ethanol helps to remove the remaining salts and SDS from the preparation.
10. Allow tube to dry for ~5 minutes. Add 50 ul TE to tube. If needed, centrifuge tube briefly to pool TE at bottom of tube. DNA is ready for use and can be stored indefinitely in the freezer.

Solutions:

Solution 1:	per 500 ml:
50 mM glucose	9 ml 50% glucose
25 mM Tris-HCl pH 8.0	12.5 ml 1 M Tris-HCl pH 8.0
10 mM EDTA pH 8.0	10 ml 0.5 M EDTA pH 8.0

Add H₂O to 500 ml.

Solution 2:	per 500 ml:
1% SDS	50 ml 10% SDS
0.2 N NaOH	100 ml 1 N NaOH

Add H₂O to 500 ml.

Solution 3:	per 500 ml:
3 M K+	300 ml 5 M Potassium Acetate
5 M Acetate	57.5 ml glacial acetic acid

Add H₂O to 500 ml.

TE	per 100 ml:
10 mM Tris-HCl pH 8.0	1 ml 1 M Tris-HCl pH 8.0
1 mM EDTA	0.5 ml 0.5 M EDTA pH 8.0

Add H₂O to 100 ml. Optional: RNAse can be added to TE at final concentration of 20 ug/ml.

 

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