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Help! Double restriction digestion failed.... - (Oct/22/2009 )

Hello Fellas...

I start my double restriciton digestion few weeks ago and now I am totally stucked.

I have cloned an insert with into a TA vector pcr2.1. I have sequenced it and confirmed that my insert is correct. My insert carry a NdeI RE site.

then i try to digest my vector with NdeI and HindIII (which is on the TA vector). I used double digestion system and i have tried several approach to optimize my plasmid amount:

1) a 50 ul reaction system:
1ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2 (NEB)
up to 50ul with water

2) a 50 ul reaction system:
2ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

3) a 50 ul reaction system:
0.5ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

I try to digest them overnight and 5 hrs, but all these gave me a very weak band or even no band after the digestion. I suspect it was due to incomplete digestion as the large band i oberve was pretty thick.

I have also tried sequential digestion as some said that the buffer may not be suitable for both enzyme. I used PCR purification to collect the first digestion during the sequential digestion then performed the second one. However, the gel result show a very weak band as the large cut plasmid and i couldnt observe my target band as it was like 300bp long.

My collegue told me to add 15ul of plasmid into a 50ul system, which is around 5ug of plasmid. I still working on that.

can anyone advice me what should i do? is that my protocol of digestion has problems? i prepare my plasmid by miniprep and yield around 300ng/ul plasmid after that.


Please advice! I am totally stucked in here right now. Thanks in advance!

-virtual-

Hello :blink:

There are some ladies around here too <_<

Anyway - have you tried a single digest with both your enzymes? ie: in one rxn cut with Nde1 and the other, HindIII? That would give you an idea if one of your enzymes and/or sites is not working.

Are you using NEB enzymes? If so, which buffer are you using for your double digest?

Clare

virtual on Oct 22 2009, 10:45 AM said:

Hello Fellas...

I start my double restriciton digestion few weeks ago and now I am totally stucked.

I have cloned an insert with into a TA vector pcr2.1. I have sequenced it and confirmed that my insert is correct. My insert carry a NdeI RE site.

then i try to digest my vector with NdeI and HindIII (which is on the TA vector). I used double digestion system and i have tried several approach to optimize my plasmid amount:

1) a 50 ul reaction system:
1ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2 (NEB)
up to 50ul with water

2) a 50 ul reaction system:
2ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

3) a 50 ul reaction system:
0.5ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

I try to digest them overnight and 5 hrs, but all these gave me a very weak band or even no band after the digestion. I suspect it was due to incomplete digestion as the large band i oberve was pretty thick.

I have also tried sequential digestion as some said that the buffer may not be suitable for both enzyme. I used PCR purification to collect the first digestion during the sequential digestion then performed the second one. However, the gel result show a very weak band as the large cut plasmid and i couldnt observe my target band as it was like 300bp long.

My collegue told me to add 15ul of plasmid into a 50ul system, which is around 5ug of plasmid. I still working on that.

can anyone advice me what should i do? is that my protocol of digestion has problems? i prepare my plasmid by miniprep and yield around 300ng/ul plasmid after that.


Please advice! I am totally stucked in here right now. Thanks in advance!

-Clare-

Hey

You should check this site for double digestions

http://www.fermentas.com/doubledigest/index.html

Maybe the ratio of a RE to another should be changed
for example:
they recommend:
Buffer R
HindIII
2-fold excess of NdeI
Incubate at 37C

Or

Buffer 2X Tango™
2-fold excess of HindIII
2-fold excess of NdeI
Incubate at 37C


OR just increase the volume of your REs
Hope this helps

-Deezoo-

virtual on Oct 22 2009, 05:45 AM said:

Hello Fellas...

I start my double restriciton digestion few weeks ago and now I am totally stucked.

I have cloned an insert with into a TA vector pcr2.1. I have sequenced it and confirmed that my insert is correct. My insert carry a NdeI RE site.

then i try to digest my vector with NdeI and HindIII (which is on the TA vector). I used double digestion system and i have tried several approach to optimize my plasmid amount:

1) a 50 ul reaction system:
1ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2 (NEB)
up to 50ul with water

2) a 50 ul reaction system:
2ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

3) a 50 ul reaction system:
0.5ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

I try to digest them overnight and 5 hrs, but all these gave me a very weak band or even no band after the digestion. I suspect it was due to incomplete digestion as the large band i oberve was pretty thick.

I have also tried sequential digestion as some said that the buffer may not be suitable for both enzyme. I used PCR purification to collect the first digestion during the sequential digestion then performed the second one. However, the gel result show a very weak band as the large cut plasmid and i couldnt observe my target band as it was like 300bp long.

My collegue told me to add 15ul of plasmid into a 50ul system, which is around 5ug of plasmid. I still working on that.

can anyone advice me what should i do? is that my protocol of digestion has problems? i prepare my plasmid by miniprep and yield around 300ng/ul plasmid after that.


Please advice! I am totally stucked in here right now. Thanks in advance!


Just a guess here, but because the conditions you described should digest the DNA no problem, I am wondering if you are sure the insert is in the CORRECT orientation to use the HindIII site on the pCR2.1 vector. With this vector and T/A cloning, the insert could be in either direction.....which means that if your NdeI site is on the side of the vector where HindIII occurs on pCR2.1, a digest will appear to only cut once even though both enzymes are working. When using the restriction sites available on the vector to recleave your clone, you have to make sure you are in the correct orientation, which usually requires screening several positive clones (sometimes more if you are unlucky :D to get one in the orientation you want. If you have other positive clones, test some DNA with the same enzymes from them....

Warren..

-Warren-

Warren on Oct 23 2009, 08:24 AM said:

virtual on Oct 22 2009, 05:45 AM said:

Hello Fellas...

I start my double restriciton digestion few weeks ago and now I am totally stucked.

I have cloned an insert with into a TA vector pcr2.1. I have sequenced it and confirmed that my insert is correct. My insert carry a NdeI RE site.

then i try to digest my vector with NdeI and HindIII (which is on the TA vector). I used double digestion system and i have tried several approach to optimize my plasmid amount:

1) a 50 ul reaction system:
1ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2 (NEB)
up to 50ul with water

2) a 50 ul reaction system:
2ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

3) a 50 ul reaction system:
0.5ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

I try to digest them overnight and 5 hrs, but all these gave me a very weak band or even no band after the digestion. I suspect it was due to incomplete digestion as the large band i oberve was pretty thick.

I have also tried sequential digestion as some said that the buffer may not be suitable for both enzyme. I used PCR purification to collect the first digestion during the sequential digestion then performed the second one. However, the gel result show a very weak band as the large cut plasmid and i couldnt observe my target band as it was like 300bp long.

My collegue told me to add 15ul of plasmid into a 50ul system, which is around 5ug of plasmid. I still working on that.

can anyone advice me what should i do? is that my protocol of digestion has problems? i prepare my plasmid by miniprep and yield around 300ng/ul plasmid after that.


Please advice! I am totally stucked in here right now. Thanks in advance!


Just a guess here, but because the conditions you described should digest the DNA no problem, I am wondering if you are sure the insert is in the CORRECT orientation to use the HindIII site on the pCR2.1 vector. With this vector and T/A cloning, the insert could be in either direction.....which means that if your NdeI site is on the side of the vector where HindIII occurs on pCR2.1, a digest will appear to only cut once even though both enzymes are working. When using the restriction sites available on the vector to recleave your clone, you have to make sure you are in the correct orientation, which usually requires screening several positive clones (sometimes more if you are unlucky :D to get one in the orientation you want. If you have other positive clones, test some DNA with the same enzymes from them....

Warren..



Dear all,

I think the orientation should be correct as I have sequenced my vector before....

anyway thanks for your kindest help, i will try to increase the RE volume and other methods suggested!

thanks for all,

Bests,
Virtual

-virtual-

Clare on Oct 22 2009, 05:57 PM said:

Hello :)

There are some ladies around here too :D

Anyway - have you tried a single digest with both your enzymes? ie: in one rxn cut with Nde1 and the other, HindIII? That would give you an idea if one of your enzymes and/or sites is not working.

Are you using NEB enzymes? If so, which buffer are you using for your double digest?

Clare

virtual on Oct 22 2009, 10:45 AM said:

Hello Fellas...

I start my double restriciton digestion few weeks ago and now I am totally stucked.

I have cloned an insert with into a TA vector pcr2.1. I have sequenced it and confirmed that my insert is correct. My insert carry a NdeI RE site.

then i try to digest my vector with NdeI and HindIII (which is on the TA vector). I used double digestion system and i have tried several approach to optimize my plasmid amount:

1) a 50 ul reaction system:
1ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2 (NEB)
up to 50ul with water

2) a 50 ul reaction system:
2ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

3) a 50 ul reaction system:
0.5ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

I try to digest them overnight and 5 hrs, but all these gave me a very weak band or even no band after the digestion. I suspect it was due to incomplete digestion as the large band i oberve was pretty thick.

I have also tried sequential digestion as some said that the buffer may not be suitable for both enzyme. I used PCR purification to collect the first digestion during the sequential digestion then performed the second one. However, the gel result show a very weak band as the large cut plasmid and i couldnt observe my target band as it was like 300bp long.

My collegue told me to add 15ul of plasmid into a 50ul system, which is around 5ug of plasmid. I still working on that.

can anyone advice me what should i do? is that my protocol of digestion has problems? i prepare my plasmid by miniprep and yield around 300ng/ul plasmid after that.


Please advice! I am totally stucked in here right now. Thanks in advance!



Dear Clare,

I didnt use single cut to confirm and now i am going to do so

I am using NEB buffer 2 for the double digestion but it didnt work....

thanks for ur advice!

Virtual

-virtual-

Deezoo on Oct 22 2009, 08:51 PM said:

Hey

You should check this site for double digestions

http://www.fermentas.com/doubledigest/index.html

Maybe the ratio of a RE to another should be changed
for example:
they recommend:
Buffer R
HindIII
2-fold excess of NdeI
Incubate at 37C

Or

Buffer 2X Tango
2-fold excess of HindIII
2-fold excess of NdeI
Incubate at 37C


OR just increase the volume of your REs
Hope this helps


Dear Deezoo,

I am using NEB product, so i duno have the buffer 2x tangos or others,
but i will try to increase the enzyme volume, thanks for ur advice!

Virtual

-virtual-