DNA shortening with Bal31 nuclease, protocol

Keywords: Zappe; DNA shortening; Bal31

Bal31 shortening

Description Related Contents

Contributor: Zappe, H

Reference: -

1. Digest 30 mg of pUC19 plus insert in a final volume of 200 ml. Check by electrophoresis for complete digestion.

2. Prepare 10 eppendorfs, marked T0 to T10, containing 75 ml TE, 1 ml glycogen and 10 ml phenol.

3. Combine 66 ml DNA from (1) (ie 10 mg), 50 ml 5X BAL-31 buffer and 133 ml H2O. Equlibrate at 37 C for 5 min then add 2 ml BAL-31 (5 U).

4. Remove 25 ml aliquots (ca 1 mg) at 1 min intervals (T=0 to T=9) into tubes prepared in (2) above.

5. Clean-up the reactions and finally resuspend in 18 ml of H2O. Add 2 ml of second enzyme buffer, 5X excess of enzyme and digest for 1 h.

6. Run gel of 5 ml of all samples to see which time intervals can be used to produce fragments of the desired size.

7. Clean-up by the reactions and finally resuspend in 20 ml TE (found that the filling-in reaction was not necessary prior to ligation).

8. Combine in a tube 4 - 8 ml insert (above), 2 ml vector (5 mg cut, genecleaned and resuspended in 30 ml TE) and water to a final volume of 10 ml. Add 10 ml 2X ligation mix (1.25 U ligase per ligation). Ligate for minimum of 2 h at room temp then transform 5 ml of the ligation to 100 ml competent cells and plate 200 ml expression mix on one X-gal plate. Should get enough colonies to find all required deletions for the particular time slot.

9. Miniprep to find shorts. Minipreps need not be digested for first screening on gels.

10. Maxiprep before sequencing.

Heinekoff shortening of DNA fragments, description

Keywords: Zappe; Heinekoff; ExoIII; shortening

Heinekoff shortening

Protocol Related Contents

Description;

Shortening of DNA using ExoIII nuclease. Insert to be shortened must have two correct types of r/e sites on side of insert to be shortened. One with a 3' overhang to protect and one that is blunt or 5' overhang to cut back on.

To see what enzymes have which overhangs, see Restriction enzyme sites. The enzymes with negative values for the overhang are protected, eg KpnI with overhang of -4.

Heinekoff shortening of DNA fragments, protocol

Keywords: Zappe; Heinekoff; ExoIII; shortening

Heinekoff shortening

Related Contents Description R/E sites

Contributor: Zappe, H

Reference: -

1. Digest 12 mg DNA with appropriate enzymes: one 5' prime overhang and one 3' to protect the vector. As the sites in the MCS are close together, cut 6 mg with each of the enzymes, check for cutting on a minigel, then mix digestions and add a little more of each enzyme (2 X excess).

2. Precipitate and resuspend in 100 ml Exo III buffer

3. Prepare 10 tubes with S1 mix (20ml per tube) and hold on ice. Label them 0, 20, 40, 60, etc. for time of treatment.

4. Equilibrate DNA at 37 C (5 min), and remove 1st 9 ml sample (T=0) to an S1 tube.

5. Add 300 U Exo III to remaining DNA, mix quickly!.

6. Remove 9 ml samples from the tube kept at 37 C at 20 s intervals to the respective S1 tubes and mix well.

7. Raise S1 mixes to room temp and leave for 30 min.

8. Add 3.5 ml S1 stop and place at 70 C for 10 min.

9. Remove 6 - 8 ml from every 2nd or 3rd fraction and run gel (0.7 % - as fragment is not released from the vector as with BAL-31 shortening) and check shortening.

10. Add 3 ml Klenow mix to each tube and 1 ml klenow enzyme and leave for 3 min at room temperature (to fill-in any sticky ends).

11. Add 1 ml dNTP's (0.125 mM/mg DNA)/tube and leave 5 min at room temperature.

12. Add 100 ml ligation mix.

13. Ligate 1 - 3 h at room temperature and transform cells eg LK111: 10 ml ligation + 50 - 100 ml cells.

14. Miniprep and restriction analysis to find resections.

Southern blots (Zappe), autoradiography

Keywords: Zappe; Southern; hybridization; DNA; autoradiography

Southern blots: autoradiography

Related Contents

Contributor: Zappe, H

Reference: Smith and Summers, (1980), Anal. Biochem. 109:123-129

Setting up the autorad.

1. After the last wash seal the filter in a plastic bag (do not let it dry!) free of air bubbles, place a film on each side in a cassette and store O/N at -70 C. Develop one film the next day: if underexposed, leave the second one another 24 h.

2. If the background is too high, remove the filter from the bag and wash again, increasing the stringency.

3. The filter can be used with a another probe if the first probe is stripped off by washing the filter for about 10 min in water at > 80 C.

Southern blots (Zappe), description

Keywords: Zappe; Southern; hybridization; DNA

Southern transfer of DNA (Zappe)

Related Contents Other Southerns

Description:

Southern transfer of DNA and blots (radioactive 32P). Harold's method.

The protocol can be sub-divided into various stages. Use the browse buttons to go through them, or click on the related topics button, or pick from below.

1. Pretreatment

2. Transfer

3. Nick translation

4. Spin column purification

5. Wash/hybing

6. Autorad

Southern blots (Zappe), nick translation

Keywords: Zappe; Southern; hybridization; DNA; nick; translation

Southern blots: Nick translation

Related Contents Southern (Zappe)

Contributor: Zappe, H

Reference: Rigby et al., 1977. J. Mol. Biol. 113:237.

Caution:

Full radiation safety protocols

Nick-translation

1. The nick-translation Amersham kit uses a 100 ml reaction and 10 ml of 32P label which is wasteful and unnecessary! The reaction can be scaled to half or to 1/4 of these volumes and still produce an excess of labelled probe.

2. Thaw the kit buffers and hold on ice. Always keep the enzyme solution on ice.

3. Combine the following in an eppendorf in order:

   		5 ml DNA  (0.3-1 mg)
   
   		5 ml kit solution 1
   
   		2.5 ml kit solution 2
   
   		10 ml water
   
   		2.5 ml 32P dN(A or C)TP
   
   

4. Incubate for 2 h at 15 C.

5. One can do TCA precipitable (Sambrook et al., Appendix E, page 18) counts on the reaction to obtain accurate % incorporation (eg. for a new kit), but normally this is not necessary.

6. Make up the volume of the nick-translation to 100 ml by adding the appropriate amount of STE

7. Remove 1 ml to a scintillation vial (eject tip into the vial) for a total count control.

8. Pass the rest of the sample through a spin coloumn, and again remove 1 ml to determine the incorporated counts. Calculate the total counts recovered and compare with the control for % incorporation. Should be 50 % or more. (incorporation can vary depending on labelled nucleotide used vs GC content of the DNA). This method is not as good as TCA's but is quick and good enough. As some of the DNA remains on the coloumn after 1 pass the % calculated is usually an underestimate.

Southern blots (Zappe), pre- treatment

Keywords: Zappe; Southern; hybridization; DNA

Southern blots: Pre- treatment

Related Contents

Contributor: Zappe, H

Reference: Smith and Summers, (1980), Anal. Biochem. 109:123-129

Pre-transfer treatment

1. Run Tris-acetate agarose gel of DNA samples over night to improve resolution. Load 1 - 5 mg total digest of chromosomal DNA per lane for homologous hybridization to clone; 100 - 200 ng per lane for plasmid to plasmid hybridization depending on the number of bands. Run l DNA markers on one of the side lanes.

2. Stain with EtBr and take a polaroid picture of the gel using type 665 Pos/Neg film: approx 80s exposure using the 254nm UV light source. This gives a very good quality print and a negative for future printing but, more importantly, the irradiation of the DNA causes breaks which aids in the more efficient transfer of larger fragments. Using a piece of paper alongside the gel, make a template of the markers. The blot will be the same size as the gel, and the size template can be used as a reference when the hybridization is complete. Alternatively, run DIG-labelled standards.

3. Soak the gel twice for 15 min in 2-3 gel volumes of depurination solution (ST1) at room temperature with gentle shaking, to partially hydrolyse the DNA by acid depurination. This causes double-stranded breaks in the DNA and aids transfer of large fragments (=20kb). Do not overdo!. Rinse with dH2O.

4. Soak the gel twice for 15 min in 2-3 gel volumes of denaturation solution (ST2) at room temperature with gentle shaking (to denature the DNA.). Rinse with dH2O

5. Soak the gel twice for 30 min in 2-3 gel volumes of neutralizing solution (ST3) at room temperature with gentle shaking (to neutralize). Rinse with dH2O.

6. Continue with either DNA transfer .

Southern blots (Zappe), spin column purification

Keywords: Zappe; Southern; hybridization; DNA; purification; recovery

Southern blots: Spin coloumn purification

Southerns DNA purification

Contributor: Zappe, H

Reference: Sambrook et al. Appendix E, p 37.

Spin coloumn purification

(removal of un-incorporated dNTP's from the probe solution)

1. The method used is exactly that described in Sambrook et al. Appendix E, p 37.

2. Use a 15 ml Falcon tube to hold the syringe.

3. Make the plug from siliconized sterile glass wool and use the plunger to position it at the bottom.

4. Do all spins at 55 % (about 1200 RPM) on the swing-out rotor for 4 min.

5. After spinning the probe through, one can wash the coloumn with 100 ml STE and recover a bit more probe, but keep this separate from the rest (the nucleotides only start coming out on the third or fourth wash).

Southern blots (Zappe), mono- directional transfer

Keywords: Zappe; Southern; hybridization; DNA; transfer

Southern blots: Mono-directional transfer

Transfer Southern

Contributor: Zappe, H

Reference: Smith and Summers, (1980), Anal. Biochem. 109:123-129

Mono-directional transfer

1. Place the gel on a piece of glass on a flat surface and dab dry with paper towel.

2. Cut off all unnecessary peices of gel and cut one corner to serve as a reference. Then cut a piece of Hybond-N+ to fit (handle only with a piece of protective paper on each side or use tweezers - never touch it!) and float it onto the ST3 solution wash to wet, then allow to soak for about 2 min.

3. Layer the membrane onto the gel being sure to eliminate all air bubbles.

4. Wet 3 pieces of Whatmans No 1 filter paper in the same solution and layer them on top of the Hybond-N+.

5. Place about 30 - 50 mm of dry paper towels on top, followed by another piece of glass and about a 2 kg weight to compress the sandwich.

6. Allow to transfer for 60 - 90 min.

7. Peel everything off, and remove the Hybond-N+ filter for alkali fixation

8. Place the membrane on 3 layers of absorbent filter paper (Whatmans 3MM) soaked in 0.4 M NaOH. The manufacturer suggests a fixing time of 2 - 60 min, but recommends 20 min.

9. Rinse the membrane briefly with 5X SSC with gentle shaking for 1 min. Seal the membrane in a plastic bag and continue with hybridisation or store at 4 C.

Southern blots (Zappe), bi- directional transfer

Keywords: Zappe; Southern; hybridization; DNA; transfer

Southern blots: Bi-directional transfer

Related Contents Southern (Zappe)

Contributor: Zappe, H

Reference: Smith and Summers, (1980), Anal. Biochem. 109:123-129

Bi-directional transfer.

1. Wet both Hybond-N+ membranes and filter paper in ST3.

2. Place 3 pieces of Whatmane No.1 filter paper on a glass plate, then a piece of Hybond- N+, followed by the gel, more Hybond-N+ and filter paper as for mono- transfer.

3. Lift this sandwich from the glass plate and place on 30mm of paper towel. Place a further stack of paper towel on top, followed by a glass plate and the weight.

4. Allow to transfer for 60 - 90 min., and then continue with alkali fixing with both membranes.

Southern blots (Zappe), hybridization and washing steps

Keywords: Zappe; denature;

Southern blots: Hybridization/washing (Zappe)

Related Contents

Contributor: Zappe, H

Reference: Both Sambrook et al. and the book "Nucleic acid Hybridzation. A Practical Approach" (edited by Hames and Higgins) discuss all aspects of hybridization - good reading!!.

Hybridization of probes to DNA bound to Hybond-N+/ Nitrocellulose filters.

1. The method is essentially the same as Sambrook et al. but with a few speeded up steps. The steps are described in Sambrook et al. page 9.47 - 9.54.

2. Make up the Prehybridization/Hybridization solution:

3. Float the filter on 6 X SSC to wet, then soak for 2 min.

4. Place the filter in a plastic bag, add 75 ml/cm2 prewarmed hybridization fluid, remove all air, seal the bag and prehybridize for 1 to 3 h, with gentle shaking at the hybridization temperature. (Calculate this temp. using the formula on pg 9.50 point 11A). Remove as much air from the bag as possible.

Denaturing the probe

1. Make a solution of probe and TE to a final vol. of 100 ml, seal the Eppendorf with tape and boil for 5 min then cool immediately on ice.

2. The alkali denaturing method of Meinkoth and Wahl (1984) (Anal. Biochem. 138:267) can also be used.

1. Cut off a corner of the bag, add the denatured probe, reseal, and hybridize with shaking at the same temp. for 16 h (O/N).

2. Add about 1 x 107 CPM for the probe.

Washing the filter after hybridization.

The stringency of the washing steps determines the sensitivity of the method. If the probe is not degenerate, then high stringency washes are used.

1. Wash twice at R/T with 2 X SSC, 0.5% SDS to rinse off unbound probe.

2. Wash for 15 min at 40 C in 0.1 X SSC, 0.1% SDS. Check the counts on the filter: Areas where no counts are expected should be clean, otherwise wash again.

3. Increase the temperature for further washes if the backgroung is a problem.