Dear Jaya

i agree with the 2 previous commentators. Generally this works. Alternatively for a better purification free of salt  (and residual complex mucopolysaccaride residues into sic to agarose) which can sometimes help with samples that fail to amplify:

• Take excised agarose slice 
• Place inside a 0.5ml eppendorf tube which has been deliberately been pierced with a needle
• So sometimes the agarose slice can be squeezed through a wide bore needle using a syringe into the inner tube or recovery facilitated by adding glass wool to this inner tube ( this was done routinely in the early 90s before the advent of purification kits):
• http://www.jcancer.org/v03p0093.htm
• place slice in pierced 0.5ml tube into a 1.5ml eppendorf 
• Spin this two tube arrangement at 13,000rpm for 10 minutes during which time your aqueous component plus DNA will make its way via the hole into the 1.5ml eppendorf
• Analogous with the above the eluate can often be used successfully in secondary PCR.  Occasionally however this is not true by virtue of excessive salt and residual mucoploydacharide so desalt the eluate by phenol chloroform extraction and ethanol ppt
• http://www.oardc.ohio-state.edu/stockingerlab/Protocols/PhenolExtraction.pdf
• Specifically 

Most of the time as indicated eluate derived directly from your gel slice will work in PCR but sometimes this additional purification will improve PCR efficiency 

Now to address what could be a potential problem based on your description: 

• An alternative approach might be to use an initial temp in PCR 2C higher to eradicate extra bands and then purify PCR reaction itself by commercial column or phenol chloroform extraction and ethanol ppt; as described
• Alternatively and/ or in addition take 1ul of the above material and reamplify: 
• Preferably reamplify with a secondary forward primer internal to the original forward primer and with a higher Tm ( Hemi nested PCR)
• This will lead to more specific secondary PCR and reduce the likelihood of re PCR resulting in a smeared product which can represent non specific plus specific amplification 
• Better still having purified PCR  product in solution or from extracted and purified material clone; make mini preps of resulting material; Anplify by PCR from different plasmid based extracts
• The advantage of this more involved approach is that you obtain abundant specific amplicon 

Wash gel slice with water, freeze it at -20 or -70 couple of times and centrifuge it at max speed in a regular microcentrifuge. The liquid above the squeezed gel can be used for re-PCR.

You don't need to freeze and thaw the agarose slice. Just add water (PCR grade!!) smash the agarose with a yellow tip, centrifuge as Michael proposes and use 1-2 microl to the next PCR

Dear Jaya

i agree with the 2 previous commentators. Generally this works. Alternatively for a better purification free of salt  (and residual complex mucopolysaccaride residues into sic to agarose) which can sometimes help with samples that fail to amplify:

• Take excised agarose slice 
• Place inside a 0.5ml eppendorf tube which has been deliberately been pierced with a needle
• So sometimes the agarose slice can be squeezed through a wide bore needle using a syringe into the inner tube or recovery facilitated by adding glass wool to this inner tube ( this was done routinely in the early 90s before the advent of purification kits):
• http://www.jcancer.org/v03p0093.htm
• place slice in pierced 0.5ml tube into a 1.5ml eppendorf 
• Spin this two tube arrangement at 13,000rpm for 10 minutes during which time your aqueous component plus DNA will make its way via the hole into the 1.5ml eppendorf
• Analogous with the above the eluate can often be used successfully in secondary PCR.  Occasionally however this is not true by virtue of excessive salt and residual mucoploydacharide so desalt the eluate by phenol chloroform extraction and ethanol ppt
• http://www.oardc.ohio-state.edu/stockingerlab/Protocols/PhenolExtraction.pdf
• Specifically 

Most of the time as indicated eluate derived directly from your gel slice will work in PCR but sometimes this additional purification will improve PCR efficiency 

Now to address what could be a potential problem based on your description: 

• An alternative approach might be to use an initial temp in PCR 2C higher to eradicate extra bands and then purify PCR reaction itself by commercial column or phenol chloroform extraction and ethanol ppt; as described
• Alternatively and/ or in addition take 1ul of the above material and reamplify: 
• Preferably reamplify with a secondary forward primer internal to the original forward primer and with a higher Tm ( Hemi nested PCR)
• This will lead to more specific secondary PCR and reduce the likelihood of re PCR resulting in a smeared product which can represent non specific plus specific amplification 
• Better still having purified PCR  product in solution or from extracted and purified material clone; make mini preps of resulting material; Anplify by PCR from different plasmid based extracts
• The advantage of this more involved approach is that you obtain abundant specific amplicon 

Thanks a lot for your suggestions.. i had previously tried what the first two commentators suggested... but still repeated it today and I am still getting some smear along with the band of interest on re-pcr. I guess if i perform a pcr again.. all i would be able to see will be a smear.

I suggest three approaches: (1) Use unrelated control primers to produce a specific band, extract, and reamplify.  In this way you can check your technique. (2) Redesign your experimental primers so that reamplification is not an issue. (3) Use TAE buffer rather than TBE

Thank you.. And I am using TAE buffer only.

Dear Jaya

• Have you tried increasing the annealing temp in your primary PCR ? 
• If you re amplify as mentioned it is common practice to use one of your primers internal to your first set of primers to minimise non specific amplification
• Failing that as I have already said you might want to go down the cloning route although admittedly this is much more work; but if all else fails
• i would start as Michael and myself have suggested with new primers and an internal ( nested) primer for secondary PCR; plus higher annealing temp
• Re PCR with a maximum of 1/100 of your starting PCR as template 
• If you continue to experience problems I would also suggest cleaning up your primary reaction by SAP EXO digestion before proceeding with secondary PCR
• I should have been more explicit about the fact that you MUST do this for nested PCR where you use an alternative second primer during your second round of PCR:
• http://www.csub.edu/~kszick_miranda/Nested%20PCR.doc

I have tried a higher annealing temperature but it didn't work.. And cloning I know is a whole lot of work.. actually my aim is to just get it sequenced.. 

hello, Dear;

There are many different methods of extracting DNA bands from an agarose gel. Which you can choose one of them depend on the consumables you have available in your lab.

first: Cutting out the DNA band

then you can one of under methods:

-Spin-columns (Nucleic acid purification columns)

-Dialysis tubing (semi-permeable membrane, Visking tubing)

-Paper strip method

-Recovery of DNA from Agarose Gels: please enter on:

http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_SourceBook_Section_VI_-_Recovery_of_DNA_from_Agarose_Gels.pdf

Hey, I have another  very simple idea if you have not tried it. I had similar problems and could not re-amplify or do cloning with my eluted PCR product. I just switched to elute the DNA from the column with ddH2O. The elution is not as efficient but it solved my problem.

Dear Jaya

now I know you wish to sequence your product then some sort of purification following any of the kit alternatives to DNA extraction is necessary 

• Thus you can purify by a plethora of kit alternative methods as suggested and then treat your PCR amplicon with sap and Exo to destroy nucleotides and primers respectively or simply extract with a kit which will remove those components and salt as well
• Since you have increased your annealing temp and that has eradicated product I think it might be safer and quicker to simply design 2 sets of alternative primers and test both. Otherwise you could be optimising your PCR for weeks
• I have sequenced 1000s of PCR amplicons in the UK and US and sometimes certain primers don't work well and the most pragmatic and quickest solution is to try alternative primers
• i should have asked: How big is your PCR product ? If under 500bp you should - with good primers - and high quality template be able to carry out 1 x PCR reaction, purify and sequence without having to perform secondary amplification with attendant non specific amplification issues
• If > 1kb -2kb the target will generally amplify less efficiently even with good quality template especially if your target sequence has > 60% GC; runs of 5 or more GC residues together or worse still if your target is > 50% AT rich
• For sequencing and efficient amplification if you are not already doing so I would recommend and routinely use a high processive hot start proof reading polymerase like NEB phusion and not Taq ( or Taq analogs) 
• https://www.thermofisher.com/order/catalog/product/F530L?s_kwcid=AL!3652!3!101843908405!p!!g!!phusion&mkwid=sChheze2S-dm_pcrid_101843908405_pkw_phusion_pmt_p_slid__&gclid=CM6fp4WRjM8CFcUp0wodpUsOlA
• https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530
• This should yield enough product from good quality template of high fidelity to directly purify and sequence without the need for secondary amplification
• If you are attempting to capture a whole gene as 1 PCR amplicon > 2kb incidentally you might be better for the purposes of sequencing to amplify in bite sized over lapping bits of ~ 500bp; purify sequence and then overlap to create a contiguous which spans your whole gene
• What is your template ? Genomic DNA or cDNA ? The quality quantity and integrity of your template will have a huge impact on PCR efficiency. I will not go into this now as this answer could turn into a discursive essay !! However I could provide advice on these issues if you post an answer to this question or send me a message 

Thanks a ton for your suggestions... the amplicon of my interest is approx. 250 bps and I have been using a master mix containing Taq polymerase.. 

http://www.sigmaaldrich.com/catalog/product/sigma/p1107?lang=en®ion=IN

Dear Jaya

for an amplicon of that size you should able to PCR with high efficiency and thus from 1 x round of PCR before purifying and sewuencing

what is more I have used that hot start ready mix from Sigma and it routinely works well 

Take a look at other answers by me and others to OCR purification for sequencing without kits

https://www.researchgate.net/post/Can_we_use_unpurified_PCR_product_for_sequencing_if_there_is_a_good_band_with_little_primer_diamer#view=56cdf59e5f7f7181668b457d

 Thanks.. I went through it.. What if I send the amplified product which has a little smear too directly for sequencing?

Hello Jaya. It isn't impossible I guess that your amplified product is smearing on account of too much loading. Is it very bright ?  

Hello Jaya,

There are two other possibilities for extracting DNA from an agarose gel slice:

1. Use low melting point agarose and TAE buffer for the gel and electrophoresis; cut out band and place in microtube, heat to 42C and degrade the agarose with beta-agarase enzyme followed by DNA precipitation (1/10 volume NaAcetate pH5 then 1.2 volumes isopropanol, freeze -20C for 30 min) and recovery by centrifugation (5 min full speed)

2. Put the agarose into a dialysis bag with a small volume of TBE or TAE (same as in gel slice). Seal bag at both ends and place in electrophoresis tank and run a current across the slice (as you would for electrophoresis. The DNA will migrate out of the gel and into the dialysis bag. Recover the fluid around the gel slice from the bag and precipitate the DNA as above. recover by centrifugation.

For sequencing grade DNA, you can wash the pellets after precipitation/centrifugation a couple of times with chilled (-20C) 70% ethanol before drying and resuspending in storage buffer (usually just water or 1/10x TE buffer).