You said "After restriction digestion, I found very faint band for my insert  while separated from pGEMT". Did you use 'double digest' or 'sequential digest'?

I checked NEB double digestion finder [ https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder ]. It says: "For these two enzyme (KpnI and SalI), double digest is not recommended. A sequential digest is required, using each enzyme in its supplied NEBuffer.". I know  that you might use different brand name's restriction enzymes, but this will tell you that their favorite buffer are different.

If you used 'double digest', this probably the reason causing 'faint' band (insert) releasing from the vector. It is better that you use 'sequential digest', meaning: cut the vector first use one enzyme, purify, and then digest using the second enzyme.

Increasing the amount of  your target insertion therefore occupied the vector site with the largest  possibility.

Hi Yuan- Yeu Yau and Huiquan Zheng,

 Thanks for your reply.

I  did double digestion following the recommendation of Therm-fisher scientific, because  we have the restriction enzymes from them. They recommended ,

 The first digestion should be performed in 1X Tango buffer (low salt concentration buffer) with 4-fold excess of KpnI. Incubate at 37°C for 1 hour.

When the first digestion is complete, add 10X concentrated Tango buffer (amount "V") to a final 2X concentration (high salt concentration buffer) and 2-fold excess of SalI. 

        V=A/8,   A - the starting volume of the reaction mixture.

Incubate at 37°C for 1 hour.

It should be easy. Try with fastDigest enzymes and purify the fragment with Gel purification kit, then ligate the target fragment with vector piece.

Hello Parvin

I am a bit confused with your questions. Did you fail to ligate your PCR product into pGEM-T vector, or fail to cut out the insert fragment from pGEM-T clone, which you have done the sequencing and ensured the insert and the cutting sites are correct? 

The following are some tips for TA cloning.

As you used pGEM-T vector, I want to remind you to add "A" at both end of PCR product if you use the DNA polymerase with proof-reading function, for example, phusion or pfu. But it is not necessary, if you use normal DNA polymerase (Taq).

On the other hand, did you use colony PCR to make sure that your insert is ligated into the pGEM-T vector? There should be M13 sequence on the pGEM-T vector. You can try to do the colony PCR with M13 primers.

If you can describe more detailed information, I am happy to give you more precise suggestions.

Cheers

Hi Liang,

  Thank you very much. The problem   is not related with pGEMT vector.  I already did all the step you mentioned with sequencing. After sequencing, then I digested  the   the pGEMT vector with KpnI and SalI to release my insert, similarly I  did it for  expression vector.  And, the problem  is, I  am not  getting enough insert, because these two restriction enzymes are not compatible in the same  buffer and not fully digested expression vector. 

Hello Parvin

As you mentioned, the problem is that the digestion condition is not optimal.

I read the product information of SalI and KpnI (Thermo). In Tango buffer, both enzymes remain only 20% activity, so you cannot obtain enough insert from your clone. I think most plasmids are still uncut.

If you cannot change to use fast digestion enzyme (also from Thermo), you have to do the sequential digestion.

In the first round, you can use more plasmid DNA, for example 2-5 ug. After digestion, please clean up the product and do the second round digestion. I think you would get more insert compared to double digestion.

For SalI, it maintains 100% activity in buffer O.

For KpnI, it maintains 100% activity in unique buffer KpnI.

The following are the manuals.

https://tools.thermofisher.com/content/sfs/manuals/MAN0012168_SalI_10_UuL_5X1500U_UG.pdf

https://tools.thermofisher.com/content/sfs/manuals/MAN0012136_KpnI_10_UuL_4000U_UG.pdf

Hope they can help you

Cheers 

Hi Liang,

 Thanks for your valuable suggestions. I am going to do now according to your  protocol.  Could you bit explain about the cleaning process after first digestion?. Normally I would do it,  just load all the reaction on gel and cut and then clean by agarose gel spin column. Is  that right way for cleaning? 

 Hello

After the first round digestion, you can take few microliter for gel running to check if the digestion is successful or not. Usually you will see a single band around 3.3 kb (pGEM-T + your insert). For the rest samples, you can directly use the spin column to make purification. Running gel is not necessary for the first digestion product, since that would greatly reduces the yield. I am not sure if your gel spin column can also be utilized for digestion product clean-up. But I think most gel spin column is also compatible for normal DNA clean-up You need to check the protocol or manual. If not, you can use conventional method to do that. The following is the protocol.

1. Add water to make the total volume become 100 ul (This volume is adjustable).

2. Add the same volume PCI (phenol:chloroform:isoamyalcohol=25:24:1), and mix well. (If you don't have PCI, you can use chloroform)

3. Centrifuge at 12,000 rpm in 4 oC for 10 min.

4. Transfer the supernatant to new tube, and add 10 ul (1/10 X volume) sodium acetate (pH5.2) and 250 ul (2.5 X volume) absolute ethanol.

5. Shortly invert the tube and centrifuge at 12,000 rpm in 4 oC for 10 min. (In this step, you can put the tube in -20 oC for few hours, that would be helpful for precipitation.)

6. Discard the supernatant and wash the pellet with 70% ethanol.

7. Centrifuge at 12,000 rpm in 4 oC for 5 min.

8. Discard the supernatant and air dry the pellet.

9. Dissolve the pellet in 30 - 50 ul water.

You can use the sample from the first digestion to carry out the second digestion. In this time, you have to run the gel to separate your insert, and extract the fragment from the agarose gel.

In above process, you will lose some DNA. Therefore, I recommend you start with 5 ug plasmid DNA. Please make sure you use enough restriction enzyme for digestion. In single reaction, the glycerol concentration should not excess 10%, or that would result in star activity or compromise the digestion efficiency.

Hope you can get good results.

Change another kit for plasmid DNA extraction, maybe too dirty to digest. It's helpful to add a positive control for solutions.

Hi Liang,

Just for update, I got enough insert  by following your  all advice's. Now just waiting to see the ligation outcome. Keep in touch. Many thanks.

Hello Everybody,

Finally I got  only one colony in my ligation plate, and I am very lucky that this one contain my insert. Thanks everybody for your valuable suggestion and special thanks to Liang for your all advice's that makes me happy today.

@ Parvin,

Good to hear that. You should also need to make sure that your gene has no base mutations by quickly sequencing it before next step (such as transformation and such).