University of Southampton
School of Biological Sciences

CLONING DNA FOR SEQUENCING


PURIFYING DNA FROM AN AGAR GEL

This describes a Quiagen based system to purify a PCR-DNA fragment from either TEB/TAE agar gels. Several commercial kits are avaiable and carry out this process successfully, e.g. Hybaid (note the PCR size fragment that these kits are able to purify before purchase).

    1. The required DNA band was cut out using a sterile blade (the dissection removed as much excess agar as necessary).

    2. The band was weighed in grams (N.B. an eppendorf is c. 900 mg).

    3. 300µl of QXI (Quiagen sys.) was added per 100mg of gel

<100 bp DNA 6 vols of QXI were added
0.1-4 kb DNA 3 vols of QXI were added
> 4 kb DNA 3 vols of QXI were added

and 2 vols of dH2O were added

    4. QXII (glass suspension) was vortexed

    5. 10µl of QXII was added to each tube, and vortexed

    6. This was was incubated at 50oC for 10 mins (and vortexed every 2 min)

    7. The preps were washed using QXI:

Samples were spun for 30 secs, removing as much of the supernatant using 1ml blue tips and 500µl of QXI was added (mix solution with pipette), the tubes were spun briefly and the remaining QXI removed.

    8. The wash was repeated.

    9. The preps were washed using PE:

Samples were spun for 30 secs, removing as much of the supernatant using 1ml blue tips and 500µl of PE was added (mix solution with pipette), the tubes were spun briefly and the remaining PE removed.

    10. The wash was repeated twice

    11. The samples were air-dried for 30 min

    12. 10 - 20µl of HPLC H2O was added to resuspend the purified DNA sample and left at R.T. for 5 min.

PHOSPHORYLATING PCR PRODUCTS

This procedure (one of the many that have been used) is only necessary where the DNA polymerase used does not 'T-tail' the PCR fragment, i.e. generate blunt-end fragments, e.g. proofreading enzymes such as Pfu, Pwo, Tli (not necessary when using Taq amplified products).

    1. 7µl of purified DNA (50 - 100 ng) was aliquoted into an 0.5ml eppendorf on ice. Samples were dessicated (in a vacuum dryer) to achieve the correct volume/concentrations etc. if necessary.

    2. 1.0µl of kinase buffer was added to the DNA aliquot.

    3. 100uM ATP (Promega as 10mM stock) was removed from -70oC and thawed and throughly mixed (ATP precipitates out of suspension on thawing) by flicking the tube bottom. 1.5µl of this was added to the reaction tube.

    4. Lastly, 0.5µl T4 Kinase (5 u: Boehringer) was added.

    5. The reaction mixtures were vortexed and spun briefly prior to being placed at 37oC for 30 min

    6. The mix was than spun down briefly, and the volume made up to 100µl using 90µl of HPLC H2O.

    7. The reaction mix were placed at 70oC for 30min to 1 h and then spun down briefly

    8. 50µl phenol and 50µl Chloroform: IAA (24:1) were then added to the eppendorf

    9. The tube was vortexed for 30 secs and spun at 14,000 rpm for 5 min

    10. The aqueous layer (upper layer) was placed in a new eppendorf and the DNA resuspended in 11µl of 3M Na Ac and 300µl 100% EtOH

    11. Precipitation was carried out on dry ice for 10 min

    12. The tubes were spun for 15 min at 13,000 rpm

    13. The precipitate was washed with 70% EtOH

    14. After spinning the samples for another 5 min at 13, 000 rpm, and removing all the excess ethanol, the DNA was air-dried for 15 min.

    15. DNA was resuspended in 6.2µl HPLC H2O

    16. 1 µl of the phorphoralated PCR fragment was loaded alongside 1 µl of a suitable plasmid (used for the ligation process) on a minigel. The mingel analysis gave approximate concentrations of both PCR fragment and plasmid. A 1:1 concentration ratio was considered appropriate, but see notes in the Ligation section.

Phosporylated DNA Plasmid
1µl DNA (~50ng)1µl (e.g. Blue script)
4µl H2O4µl H2O
2µl Loading Dye 2µl Loading Dye
7µl final vol7µl final vol

Lambda Hind III marker was used as a molecular marker and the gel was run at 100v for 30 min.

LIGATION

Ligation is the process where a DNA fragment is inserted into an appropriate plasmid. The plasmid has been manufactured commercially to include some form of signal that indicates when DNA has been inserted into the plasmid's ring of DNA.

For example, most plasmids have been engineered to contain a multiple cloning site (MCS) which has a lac-Z gene attatched. When inserted into a suitable bacterial cell a functional MCS (Lac-Z gene) will produce beta-glactosidase which produces blue colonies. Therefore, if DNA (the fragment being incorporated) ligates successfully into the MCS, the lac-Z gene is disrupted, and the host bacteria appear as white colonies (POSITIVES).

In this subsection the plasmid (blue script, pGEM-T, pGEM-T Easy etc.) are referred to as VECTORS. The example quoted here utilises the Promega protocol with pGEM-T Easy. The purified DNA fragment is synonymous with the word 'INSERT'.

In general a 3:1 to 2:1 (vector:insert) ratio is recommended for the ligation reaction. For different size inserts, the concentration of the insert when using the standard vector of 3 kb and a 100 ng is calculated using the following formula:

vector (ng) x insert (kb)
vector (kb)		 xratio of insert
vector		 = insert ( ? ng)

e.g. Using an insert (0.5 kb) and a vector (3.0 kb: 100 ng), and a vector:insert ratio 1:3, the insert concentration (ng) will be:

vector (100ng) x insert (0.5kb)
vector (3.0kb) 		x3
1		 = insert 50 ng



However, when ligating new material for the first time, various vector : insert DNA ratios are recommended along with several control measures to find optimal cloning conditions for use with your fragment, e.g.

100 ng Vector + control DNA from manufacterer (positive control) (to check reaction)
100 ng Vector + HPLC H2O(negative control) (to check for contamination of vector solution)
100 ng Vector + c. 10 ng insert DNA
100 ng Vector + c. 20 ng insert DNA
100 ng Vector + c. 50 ng insert DNA
100 ng Vector + c. 100 ng insert DNA

The DNA ligation procedure was carried out as follows:

    1. The following components of the Promega ligase kit were brought down from -70oC storage, vector DNA, ligase buffer, T4 DNA ligase and placed on ice and added together in the following order:

1x reaction mixConcentrations
HPLC-grade H2Oup to 10.0µl -
10x ligase Buffer 2.0µl -
vector 1.0µl
DNA insert 4.0µlc. 100ng
T4 DNA ligase1 (see note before adding)1.0µl5 u/µl
Final Volume, Master Mix10.0µl

a) Equal repeatability was achieved by scaling down the reactions by adding half the volumes per reaction (i.e., 5.0 µl total volume)

    2. (10µl final vol.) was made up with HPLC H2O

    3. 2 µl 10 x ligase buffer (throughly shaken to resuspend ATP granules) was added

    4. 1 µl vector (100ng) was added

    5. 4 µl (total 100ng) of insert (e.g. 2.0 kb fragment) was placed in a 0.5 ml eppendorf.

    6. 1µl T4 DNA ligase (Promega) was added last.

    7. The reagents were vortexed and spun briefly and placed at 4oC over night (16 h, minnimum 3h).

    8. The same volumes were used for both positive and negative control reactions (adjusting volumes with HPLC H2O.

PREPARATION OF LB-AGAR AND SOC


LB-AGAR

LB-agar is the medium on which the bacterial culture used in the TRANSFORMATION section are plated. It is usualy best prepared fresh on the day the ligation is carried out, although it can be stored for several months in a suitable sealed container.

N.B. Mention of "heat sterilisation" refers to brief passing of the petri lid etc, over a yellow bunsen flame.

    1. The LB medium:

g/1000ml g/500 ml g/200 ml:
Lab M-tryptone 10.0g 5.0g 2.0g
Yeast extract 5.0g 2.5g 1.0g:
NaCl 5.0g 2.5g 2.0g:

Adjust pH to 7.0 with NaOH

2. Agar 15.0g7.5g 3.7g:

Agar (Agar bacteriological I) was added prior to autoclaving (the solution was then labelled as LB-agar).

3. The medium was allowed to cool to 50oC before adding ampicillin.

4. 50 µl Ampicillin (100mg/ml; dissolved in HPLC H2O, filter purified and aliquoted into 1.5 ml eppendorfs) was added to a sterile Falcon 50 ml tube. (N.B. Powdered ampicillin was stored at 4oC and aqueous samples stored at -20oC).

5. 50 ml LB-agar was added to falcon tubes with ampicillin.

6. The mixture was poured into sterile petri dishes (and was suitable to completely cover up to 4 petri dishes, i.e. 1 l of solution should make up to 80 petri dishes)

7. Any unused ampicillin was stored in a freezer and the LB-agar bottles were heat sealed and can be stored on the shelf for several months, but these are always inspected for fungal or other contaminants before use. The petri dishes were stored at 4o C until needed.

SOC

SOC medium is the solution in which the transformed cells are grown up prior to extracting in mini or midi preps.

    1. The SOC medium:

g/100mlg/50 ml
Lab M-tryptone2.0g 1.0g
Yeast extract 0.5g0.25g
1M NaCl1.0 ml 0.5 ml
1M KCl 1.0 ml 0.5 ml
2M Mg2+ 1.0 ml 0.5 ml
2M glucose 1.0 ml0.5 ml

    2. The Lab M-tryptone, Yeast extract, NaCl and KCl were added to 97 ml ddH2O and stirred to dissolve.

    3. The solution was than autoclaved and allowed to cool to RT.

    4. The 2M Mg2+ (1M MgCl2.6H2O/1M MgSO4.7 H2O: filter sterilised) and 2M glucose were added to the solution and the pH adjusted to 7.0 with NaOH.

    5. The complete medium was filter sterilised prior to use and stored at RT.

TRANSFORMATION

Transformation is the process where the vector is inserted into a suitable bacterial cell, 'COMPETENT CELL' or bug, e.g. a modified E.coli strain, the example used here refering to JM109 High efficiency Competent Cells (Promega).

    1. 2µl (4 µl if reaction volumes are halved) of the ligation preparation from the LIGATION section (insert/vector prep) was placed in a 1.5 ml eppendorf on ice

    2. The competent cells were removed from -70oC and thawed on ice for 5 min (N.B. since the competent cells, "bugs", are fragile, these cells are pretreated and the salt concentration maintains a porous cell surface to which the plasmid will adhere.) Do not shake or stir vigourously!!

    3. 50µl of the competent cell solution was used per 2µl ligation preparation. This was gently aliquoted and pipette mixed once, keeping on ice at all times.

    4. After all the experimental inserts had been dealt with, control transformations were also carried out using the "cont. ligation" kit-insert, and secondly with intact plasmid.

    5. The eppendorfs were left on ice for 20 min and waterbath or oven heated to 42o C exactly for next step.

    6. The eppendorfs were then placed at 42o C for 45-50 secs . The timing is crucial!. This is the 'heat-shock' treatment of the cell-mixture.

    7. The tubes were placed back immediately on ice for 2 min.

    8. The samples were taken off ice and 950µl of SOC were added to each tube.

    9. The tubes were gently inverted and placed on a slow mill or shaker at 37oC for 90 min.

    1. During this period the LB-agar/ampicillin petri dishes were removed from 4oC.

    2. 20µl X-gal (100mg/ml: stored at -20oC) was pipetted to the middle of the sterile petri dishes (work with 7 petri dishes at a time to reduce the chance of solutions drying before being spread)

    3. 100µl IPTG (100mM: Mw 238.31) was added on the X-gal drop and the two solutions spread over the plate using 100% ETOH/flame sterlised glass spreader (glass pipette adapted as a spreader, by producing a R-hand bend over a bunsen flame, and sealing the tip by melting it). N.B. the glass spreader was cooled on the moist agar before spreading the drops.

    4. The plate lids were heat sterilised and shut prior to allowing to stand for 30 min before further use.

    1. After incubation at 37o C for 90 min, the transformed competent cell cultures were gently inverted and 50µl were aliquoted to individual X-gal/IPTG/ampicillin/LB-agar petri dishes (just prepared).

    2. The glass spreader was sterilised by alcohol flaming between each application and cooled by placing on moist agar. This was then used to spread the drop across the plate throughly. Flame treat the spreader between each preparation.
    N.B. disposable plastic spreaders are also available e.g. from BDH (http://www.BDH.com).

    3. The petri lids were heat sterilised shut and left for 30 min

    4. The petri dishes were then inverted and placed at 37oC over night (12-16 h)

    5. After the o/n step the samples were removed from the oven and placed at 4oC and screened for positive cells: white colonies.

PICKING POSITIVE COLONIES

    1. White colonies were screened for by looking for the best white colony representatives (these being those that had further satellite colonies around them)

    2. A 50ml:50µl LB:Ampicillin was made up in a sterile 50 ml Falcon tube.

    3. Single positive colonies were picked using "COPAN" loops. (sterile plastic devices (lengths) with a small looped end). The striaght end was gently inserted into the chosen colony and transferred to a 3 ml aliquot of the LB:Ampicillin aliquoted into a 12 ml Falcon tube (round bottomed loose capped sterile culture tube), using a twisiting action. 3. The tubes were placed at 37o C o/n

SNAP FREEZING

After incubation 0.85 ml of the solution was mixed with 0.15 ml of glycerol, vortexed and then stored at
-21oC (this snap-frozen sample will keep for long periods in storage).

ALKALINE LYSIS

Alkaline lysis of mini or medi (depending on the size of the cultures) preparations involves the breaking down of bacterial cells to release the plasmid DNA and further purifies the plasmid DNA for the sequencing reaction. Various kits are now available that allow you to carry out this process in microspin columns. The example used here is with Promega's WIZARD-PLUS system.

    1. The remainder of the solution in the falcon tubes was spun down at 3,000 rpm for 5 min.

    2. The supernatant was decanted and all the LB-agar was removed by pipetting if necessary.

    3. 250 µl of WIZARD PLUS SV cell suspension solution were added to the tubes and these were vortexed, until all the precipitate was resuspended.

    4. A set of 1.5 ml tubes were labelled corresponding to the sample labels.

    5. The resuspended culture was aliquoted into the labelled tubes.

    6. 250 µl of cell lysis mixture was added to each tube and the tubes were inverted four times.

    7. These were left to stand for 2-3 min until the mixture cleared.

    8. 10 µl of alkaline protease were added next and the tubes were inverted four times, slowly.

    9. The tubes were left to stand for 5 min.

    9. 350 µl of neutralisation solution was added next, and the tubes inverted four times.

    10. The solution was spun at 14,000 rpm for 10 min.

    11. The supernatent was transfered to a labelled WIZARD PLUS spin column.

    12. The columns (placed as described by the manufacturer) were spun* for 1 min and the filtrate discarded.

    13. 750 µl of wash solution was added to the column, and the tubes were spun again for 1 min (the filterate discarded).

    14. 250 µl of wash solution was added and the column spun for 2 min and the filtrate discarded.

    15. A new eppendorf tube was used to replace the one in use with the spin column and 70µl of nuclease free water was added to the column.

    16. The new tube-columns were spun for 1 min.

    17. The concentration of the plasmid was tested for using a mini agarose gel, tests carried out as described in DNA QUANTITATION.

* All spins mentioned were carried out at 14,000 rpm.

TEST FOR INSERTION

Although the white colonies that were picked should have the required DNA insertion present, this is not a guarantee. Most colleagues carry out a simple test to see the size of the insert that had been cloned and amplified by transformation prior to sequencing. Two such tests are described here.

Digest method

1. A digestion reaction was carried out using the most appropriate enzymes to cut out the insertion site (plasmid) that was used. We used either of Nco I or Spl and Not I I. The reaction volumes were as follows or after manufacturer's recommendations:

    5.5 µl H20 (Tot vol 10.0 µl)
    1 µl Reaction buffer
    0.5 µl Nco I or Spl I
    0.5 µl Not I
    0.5 µl RNAse
    2 µl plasmid DNA

2. These were incubated for 2 h at 37oC

3. The restriction reaction was stopped using 1µl stop mix (0.5M EDTA/glycerol/bromophenol blue)

4. And the restrictions were run on a 0.75-1.0% gel with suitable size markers.

PCR method

Using this method, a colony can be tested for a 'true-insertion' prior to transformation.

    1. Positive (white colonies) were selected from the plates.

    2. A PCR reaction was set up:

The PCR reaction volumes were:

H2O* 46.10µl
10x Taq Reaction Buffer   2.50µl
DNTPs   0.50µl
Primer #1 (25pM)   0.50µl1ng/µl
Primer #2 (25pM)   0.50µl1ng/µl
Taq   0.40µl2.0 units
Final Volume50.00µl

#1 T7 primer sequence: 5'-CCTATAGTGAGTCGGTATTA-3'

#2 SP6 primer sequence: 5'-TAGGTGACACTATAGAATAC-3'

    3.These were picked using the COPAN looped-picker and briefly dipped, under the mineral oil layer (25 µl) of a pre-mixed PCR reaction aliquot in an 0.5 ml PCR eppendorf, and twisted in a 3ml aliquot of LB:Ampicillin (50ml:50ml) placed in a 12ml Falcon loose capped tube (see transformation).

    4. A positive and negative control was always set up with the PCR reactions.

    The PCR-program was
    (1 cycle)94o C for 4 min

    (35 cycles){94oC for 30 sec
    {50oC for 30 sec
    {70oC for 1 min (increase if > 2.0 kb)

    (1 cycle) 72oC for 6 min (hold at RT)

    5. The PCR reactions were run on a 0.75 -1% gel, and inserts sizes checked; if no insert was present 50 bp primer-diamers were noted.




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