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More colonies fron vector only control?


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#1 Bob Lu

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Posted 06 August 2010 - 07:11 AM

"from". Can't spell this morning :(

---

Maybe is it better to post the question in Molecular Cloning section?

Sorry I am sort of new here. (I am a long time reader of the archived posts, tho :P)

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I am trying to clone a (~0.8k bp) fragment from a TA construct to another expression vector (pCS2+).

The fragment is flanked by EcoRI and BamHI sites, both are unique on the TA construct and pCS2+.

I double digested the TA construct and the pCS2+ vector side by side for four hours. Then pCS2+ was CIP'd for 1 extra hour.
(I don't think CIP is really needed for double digestion?)

On gel I can see the fragment I want was released from the TA construct, so I assume the pCS2+ double digest also worked find. (And compare with the undigested pCS2+, there was a band shift.)

After gel extraction, I combined 100ng of the insert and 100ng of the vector (mole conc. should be about 3:1 ) for ligation.

For control, I just used water instead of the insert.

I used Takara kit ver 2.1, 16C O/N.

I used half of the reaction for transformation. I got about 1.5 times of colonies from the vector only control compare with the vector + insert group.

The same thing had hppened several times before for me. Sometimes I tried to still pick some colonies for PCR but almost never got any positive hit. (Got one from nearly a hundred PCR once.)

Any suggestion? Did I miss anything?

---

Some thoughts:

1. I didn't heat inactivate CIP. I heard that even with gel extration, not doing heat inactivation can still reduce ligation efficiency. However I don't see how this can make no insert control do better than the vector + insert group.

2. My vector and insert are in Tris buffer. So for control I also should have used Tris buffer. However the Takara kit handbook actually says the kit like Tris buffer better.

3. I am thinking single digested vector plus incomplete CIP is th best answer? If it is the case, without insert the single digested, not CIP'd vector can self-ligate. When (a lot of) insert was added, such vector ligates with one end of the insert but is unable to ligate with the other end (because the vector is only single digested). Does it make some sense?

Edited by Bob Lu, 06 August 2010 - 07:35 AM.


#2 NemomeN007

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Posted 10 August 2010 - 07:43 AM

"from". Can't spell this morning :(

---

Maybe is it better to post the question in Molecular Cloning section?

Sorry I am sort of new here. (I am a long time reader of the archived posts, tho :P)

---

I am trying to clone a (~0.8k bp) fragment from a TA construct to another expression vector (pCS2+).

The fragment is flanked by EcoRI and BamHI sites, both are unique on the TA construct and pCS2+.

I double digested the TA construct and the pCS2+ vector side by side for four hours. Then pCS2+ was CIP'd for 1 extra hour.
(I don't think CIP is really needed for double digestion?)

On gel I can see the fragment I want was released from the TA construct, so I assume the pCS2+ double digest also worked find. (And compare with the undigested pCS2+, there was a band shift.)

After gel extraction, I combined 100ng of the insert and 100ng of the vector (mole conc. should be about 3:1 ) for ligation.

For control, I just used water instead of the insert.

I used Takara kit ver 2.1, 16C O/N.

I used half of the reaction for transformation. I got about 1.5 times of colonies from the vector only control compare with the vector + insert group.

The same thing had hppened several times before for me. Sometimes I tried to still pick some colonies for PCR but almost never got any positive hit. (Got one from nearly a hundred PCR once.)

Any suggestion? Did I miss anything?

---

Some thoughts:

1. I didn't heat inactivate CIP. I heard that even with gel extration, not doing heat inactivation can still reduce ligation efficiency. However I don't see how this can make no insert control do better than the vector + insert group.

2. My vector and insert are in Tris buffer. So for control I also should have used Tris buffer. However the Takara kit handbook actually says the kit like Tris buffer better.

3. I am thinking single digested vector plus incomplete CIP is th best answer? If it is the case, without insert the single digested, not CIP'd vector can self-ligate. When (a lot of) insert was added, such vector ligates with one end of the insert but is unable to ligate with the other end (because the vector is only single digested). Does it make some sense?


Sounds like your Bam enzyme is going bad...try using fresh Bam... usually see these kinds of things when one enzyme is cutting better than the other. While your insert is coming out in your TA vector (even 50% is good enough), your pCS2 is not being efficiently cut by both enzymes and this will seriously affect ligation/transformation cloning.




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