9 replies to this topic

[Huongnguyen.miss]

I want to expression a gene from actinomycete. My gene's size is around 800 bp. The Restriction enzymes are BamHI and HindIII. I isolated and purified my gene and ligated into pET28 ( pEt28 was digested by BamHI and HindIII too), then i transformed in XL1blue, kept plate overnight. I got around 30 colonies, i selected 10 and isolated, digested but i could not get any band of gene. i have repeated 2 times, but all are same. 

Could someone know what is my problem? please help me, im waiting for your answers. Thank you so much. My english is not good, im so awkward,  plz don't mind ^^

[phage434]

Likely your insert has not been cut correctly. How is the insert prepared? Are the BamHI and HindIII sites on  your primer? Are there extra 5' bases before the restriction site? Are you purifying your PCR product before cutting it?

[Huongnguyen.miss]

yes.  i did. my insert was prepared by PCR with BamHI and HindIII in C-terminal and N-terminal, respectively. and cutting and purified.

i had 2 extra base before restriction site. i sent to company to check the sequencing of my gene, and all are correct.

So, how to do ((

[phage434]

Two bases is probably sufficient, but more would be prudent.

You need to make absolutely certain you purify your PCR product BEFORE cutting (and possibly, although less importantly, after). PCR enzymes will extend cut sites and block ligation unless removed prior to RE digestion.

BamHI cannot be heat killed, so you will have to purify the restriction digest with a column or other technique.

 

You can test the quality of your insert by doing a PCR reaction and cutting (in two separate tubes) with BamHI and with HindIII. Each of those products should be purified (you can heat kill the HindIII digestion). Then, ligate with that part alone (no vector). When you run the product on a gel, you should see a double length fragment corresponding to the ligation of the PCR reaction product to itself. If not, you have a problem which needs to be fixed.

 

You can have similar issues with your vector, but it is more difficult to easily test. Are you removing a sufficiently large piece of DNA from your vector that you can distinguish the singly from the doubly cut vector? You need to verify that both sites are being cut.

[doxorubicin]

It also can be useful to perform PCR on the ligation prior to transformation to see if the ligation worked at all.  There are companies you can pay to do this using a gene synthesis approach for a couple hundred dollars if you know what the sequence is.

[Huongnguyen.miss]

" Two bases is probably sufficient, but more would be prudent.

You need to make absolutely certain you purify your PCR product BEFORE cutting (and possibly, although less importantly, after). PCR enzymes will extend cut sites and block ligation unless removed prior to RE digestion.

BamHI cannot be heat killed, so you will have to purify the restriction digest with a column or other technique.

 

You can test the quality of your insert by doing a PCR reaction and cutting (in two separate tubes) with BamHI and with HindIII. Each of those products should be purified (you can heat kill the HindIII digestion). Then, ligate with that part alone (no vector). When you run the product on a gel, you should see a double length fragment corresponding to the ligation of the PCR reaction product to itself. If not, you have a problem which needs to be fixed.

 

You can have similar issues with your vector, but it is more difficult to easily test. Are you removing a sufficiently large piece of DNA from your vector that you can distinguish the singly from the doubly cut vector? You need to verify that both sites are being cut ".

thank guys for your suggestions. But im still stuck...i did transformation triplicate, but till now still i got no positive result.

And  i could not get your point 

phage434. 

test the quality of my insert by doing PCR mean after i ligate ( 16 degrre C, overnight)  , and then i do PCR? or after transformation, isolate plasmid and do PCR?

cutting in two separate tube by BamHI and HindIII, is this single digest?

[phage434]

The idea is to prepare your insert in the normal way (PCR with primers containing the HindIII and BamHI sites). Purify it. Then, in two separate reactions, cut with HindIII in one, and with BamHI in the other. Purify (or heat kill) the enzymes. Then, ligate the purified product.  You should be able to ligate the cut ends together (HindIII site to HindIII site) and (BamHI site and BamHI site). These two ligations can then be run on a gel along with the uncut PCR product. You should see a band in the enzyme cut/ligated lanes which is twice the length of the original PCR product. This will show you that each of the ends has been cut successfully and can be ligated.

[Huongnguyen.miss]

you mean that the PCR product will be purified with BamHI and HindIII, and then cut with HindIII is the first reaction and in the second reaction, i use purified product cut by HindIII and cut with BamHI ? But how about these reaction? how much volume of enzyme use ? could u give me more detail about that? 

now im trying to do  PCR, after i ligated gene into pET28 , to check gene has already inserted into vector or not. Is it ok?

im looking forward for your suggestion ...

[phage434]

I don't seem to be able to explain this well.  Let me be precise.

 

Step 1. PCR your fragment with primers containing your restriction sites

Step 2. Purify your PCR fragment (column, gel, or other technique)

Step 3. Split your purified DNA into three parts A, B, C

Step 4: Cut part A with HindIII

Step 5: Purify part A (heat kill or column). Save as A1.

Step 6: Ligate A1 to itself. Save as A2

Step 7: Cut part B with BamHI

Step 8: Purify part B with column (heat kill will not work). Save as B1

Step 9: Ligate B1 to itself. Save as B2

Step 10: Run a  gel with samples C, A2, B2

Step 11: look for a band in lanes for A2 and B2 showing a fragment of size double the length of band in lane C

Step 12: If you see no double length band in lane A2, your HindIII enzyme (or the primer creating that site) is not working

Step 13: If you see no double length bnad in lane B2, your BamHI enzyme (or the primer creating that site) is not working

 

None of this helps make your construct: it only helps you understand why other things are not working.

[Huongnguyen.miss]

thank you so much for your explanation. Its so detail. I will try to do as you told me.  and let  you know if i get the results.