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Protocols Protocols

IN-Gel Hybridization procedure. (ref. Dionne and Wellinger,PNAS 1996, 93,13902): Our favorite protocol for telomeric single stranded DNA detection.

A) Gel treatments

  After running gel (0.5-0.75% in 1xTBE buffer), stain with EtBr and photograph.   If this is a telomere blot, you should have digested and loaded at least 3  microgrammes of genomic DNA per lane.    - Immerse gel in 2xSSC for 30 at RT.  - Put gel upside down (ie the open wells down) on 2 layers of Whatman paper and cover     with Saran wrap  - Dry the gel on the gel dryer at RT (we use a BIO-RAD 583 dryer)  	first 12-14 min., stop vacuum by switching valve on pump (but let pump running)  	flip gel over  	dry another 12-14 min at RT.  
DO NOT EXCEED 30 min of drying.
	  Gel should now be very thin and even.  - Upon removal, it should stick to the saran wrap, if not just slightly wet the Whatman     papers with 2xSSC and then remove gel.    - If this is for a non-denaturing gel, put the gel now into a sealable plastic     bag and go directly to hybridization!!    - Add ~5 ml of Hybridization soln. without probe to check integrity of bag.  - Add ~20 ml of Hybridization soln. with the hot probe (see below) and seal bag     WITHOUT bubbles!  - Hybridize at 30 oC- 37 oC, overnight.    Hybridization soln:  	25 ml of 20xSSC  	1 ml 10x P-wash  (10x P-wash= 5mM Ppi (inorganic pyorphosphate), 100mM Na2HPO4)  	10 ml 50x Denhardts (or 5 ml 100x Denhardts)  	40 microliters 0.1 mM ATP  	40 microliters denatured salmon sperm DNA (50mg/ml)  To 100 ml with sterile H2O.  
B) Rehybridization of gel after denaturation (detection of all DNA)
  After appropriate exposures of the non-denatured gels are obtained, the DNA in the gel   can be denatured and detected by rehybridization.    - Put gel into a glass tray and treat with 500 ml denaturing solution for 25 min at RT  	Denaturing sln:	150 mM NaCl  			0.5 M NaOH    - remove denaturing soln and add 500 ml Neutralizing soln. Gently shake at RT for    20 min.  	Neutralizing soln:	150 mM NaCl  				0.5 M Tris.HCl pH 8.0    - Remove gel and put into a sealable plastic bag.  - Add ~5 ml of Hybridization soln without probe to check integrity of bag.  - Add ~20 ml of Hybridization soln with the hot probe and seal bag WITHOUT bubbles!  - Hybridize at 30 oC-37 oC, overnight.    
C) Probes and washings
    - Use any oligo (ours are usually 20-25mers), gel purified and which is at     100ng/microliters  - Label 100ng in a final volume of 20 microliters, 1XPNK buffer, 1 microliter PNK     and 6 microliters gamma-ATP.  - 45 min at 37 oC  - 10 min at 68 oC  - add	80 microliters H2O  	125 microliters 7.5 M NH4Acetate  	530 microliters EtOH abs. at -20 oC  - mix well, -20 oC for >1 hr.  - Spin at max speed for 45 min at 4 oC, wash once with cold 70% EtOH, respin     15 min, dry, resuspend in 30 microliters TE.  - if you count 1 microliter of this, you should get around 100 000-200 000 cpm.  - For a regular gel, you will need about half of this probe in about 25 ml (see above)      - Washes 	2x for about 1.5 hrs at room temp.  		1x for about 2 hrs at 30 oC (this step is optional, depends on                  signal to noise ratio..)    Wash solution: 0.25X SSC    - Expose appropriately (usually requires 1-2 days). If you have excess ss DNA,     a few hours to ON should be OK.  
D) Controls
  It is very useful to run ssDNA and dsDNA controls in parallel lanes. Usually, you   will have a plasmid with the target sequence cloned available. If this is the case,   digest some of this plasmid with a restriction enzyme that linearizes the plasmid.   For the dsDNA control, load about 1-5ng of this digested plasmid. For the ssDNA  control, load the same amount of the linearized plasmid but heat denature the DNA  prior to loading (10 min, 95 oC).  
E) Special Notes:
  The critical step in this procedure is the gel-drying step. You might have to   fiddle around with your gel-dryer setup to get the conditions right. After drying,   the gel should be very thin (a bit thicker than 3MM paper; but not as thin as   Saran wrap). Should the gel reswell during hybridization, then it was not dried   enough. When gels are overdried, DNA smaller than ~1.5 kb tends to be blotted out   and will end up on your 3MM support instead of staying in the gel. Detection of   such DNA in the gel is obviously rather difficult...  
With compliments from the Wellinger lab for your excellent choice of methods!!

Troubleshooting

Here are some typical problems that you might encounter when using this technique. As a free service to the community, we also suggest some ways to resolve them.


Case #1: Spotted gel (kindly provided by Julie)


Our interpretation: The spots are caused by aggregation of probe in the gel matrix. Cleaning your probe should resolve the problem for the next gel. It is almost impossible to get rid of such spots and the gel usually has to be declared a total loss! Remember: this is a non-denaturing procedure, you cannot simply heat wash or alkali treat to get rid of the problem.

X-File interpretation: Your gel has been infected by an unknown dangerous virus. Run for your life!

Alternatively: It's Michel's fault.

Seriously now, it is almost impossible to get rid completely of such spots. However, rewashing the gel at a slightly higher temperature might greatly improve the result. As an example, the same gel shown above, rewashed at 30 oC for 2:30 h in 0.25X SSC.

Case #2: Overdried gel (kindly provided by Michel)


Our interpretation: When gels are overdried, DNA smaller than ~1.5 kb tends to be blotted out and will end up on your 3MM support instead of staying in the gel. This problem is obvious in the ladder lane (lane 1). The 0.5 and 1 kb bands have been blotted out. Consequently, the ~1 kb XhoI TRF (terminal restriction fragment) cannot be detected. (For the connaisseurs of the matter: this DNA was derived from a ku- strain, thus the shorter TRFs).

X-File interpretation: The TRF has been kidnapped by an alien-life form, specialized in making life of telomere researchers miserable.

Alternatively: It's Michel's fault.

Seriously now, you might have to fiddle around with your gel-dryer setup to get the conditions right. After drying, the gel should be very thin (a bit thicker than 3MM paper; but not as thin as Saran wrap). As an example, the same DNA has been analyzed on a gel dried appropriately. Note the 0.5 and 1 kb bands in the ladder lane.

Case #3: Heavy background (kindly provided; sorry, I promised to keep the secret)


Our interpretation: The gel reswelled during hybridization, because it was not dried enough. Consequently there is excess probe all over the gel giving it a hazy (in severe cases, all black) background.

X-File interpretation: Those dammed aliens once again.

Alternatively: It's Michel's fault.

Seriously now, you can try to re-dry and reprobe the gel (but dont forget; do not overdry it!). Alternatively, one or two freeze-thaws of the gel will push the water out. Once you get rid of the excess water a quick wash (1h, 23 oC, 0.25X SSC) and a re-exposition should do the job. Shown next, the same gel after we did the freeze-thaw procedure to it.