Hi there!

If you are getting almost pure PCR product then you can use a PCR clean up kit (a column based kit) which removes the residual components of the PCR reaction mix (majorly polymerase, excess dNTPs etc.).  You can directly proceed with the restriction digestion with the elute. After restriction digestion you can perform a phenol-chloroform extraction to remove the restriction enzyme and use it for further processes. 

But if you are afraid that your insert might not be pure, then at one point either after PCR or after digestion you have to run it on an agarose gel and purify it. It is better to do it after PCR because you can set up more PCR to start with high amount so that you yield enough to proceed further. There are many ways to improve the gel elution yield. like-

1. Use lower percentage of gel

2. keep the dissolved gel in column for 2 mins before spinning (allow some time before binding).

3. wash the column twice, also keep the wash buffer in the column for 2 mins before spinning. Give an empty spin.

4. Dry the column at 65 to remove all the ethanol

5. Heat the elution buffer at 65 and keep it in column for at least 5 mins before spinning. 

There are many such tricks following which you can substantially improve your yield. You can go through the following links if you need.

Hope it helps!

https://www.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ug

http://bitesizebio.com/13508/5-more-tips-for-dna-gel-extraction/

http://bitesizebio.com/13506/10-tips-for-better-dna-gel-extraction-results/

https://www.neb.com/tools-and-resources/usage-guidelines/six-tips-for-a-perfect-gel-extraction

Hi there!

If you are getting almost pure PCR product then you can use a PCR clean up kit (a column based kit) which removes the residual components of the PCR reaction mix (majorly polymerase, excess dNTPs etc.).  You can directly proceed with the restriction digestion with the elute. After restriction digestion you can perform a phenol-chloroform extraction to remove the restriction enzyme and use it for further processes. 

But if you are afraid that your insert might not be pure, then at one point either after PCR or after digestion you have to run it on an agarose gel and purify it. It is better to do it after PCR because you can set up more PCR to start with high amount so that you yield enough to proceed further. There are many ways to improve the gel elution yield. like-

1. Use lower percentage of gel

2. keep the dissolved gel in column for 2 mins before spinning (allow some time before binding).

3. wash the column twice, also keep the wash buffer in the column for 2 mins before spinning. Give an empty spin.

4. Dry the column at 65 to remove all the ethanol

5. Heat the elution buffer at 65 and keep it in column for at least 5 mins before spinning. 

There are many such tricks following which you can substantially improve your yield. You can go through the following links if you need.

Hope it helps!

https://www.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ug

http://bitesizebio.com/13508/5-more-tips-for-dna-gel-extraction/

http://bitesizebio.com/13506/10-tips-for-better-dna-gel-extraction-results/

https://www.neb.com/tools-and-resources/usage-guidelines/six-tips-for-a-perfect-gel-extraction

Very useful! Thanks a lot for your answer!

Hi there,

Take  5ul of your pcr product and run on agarose to see if you don't have side products. If you have a single band, you can purify the other reactions directly without running on an agarose gel.

Hope this helps.