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Low labeling


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#1 Idit

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Posted 02 March 2010 - 02:31 AM

Hi all,

I'm doing indirect labeling (Aminoallyl + Cy3/5) for a couple of years. I used to get 250-500 pMol of each color with frequency of incorporation (FOI) 10+. Lately I started getting 60-100 pMol with FOI 2-6.

My prorocol is in brief:
1. Amplification with klenow, aminoallyl mix, and random primers ON.
2. The next day I clean with microcon
3. Coupling with the Cy dye in MaHCO3 0.1M buffer for 1.25h. I'm using a fresh buffer each time at pH 8.6.
4. Cleaning with Quiagen purification column.

I tried changing the Aminoallyl stock and the batch of the Cy dye, the klenow enzyme, the random primers. Does anyone have an idea what could be interrupting?

Thanks,
Idit

#2 Clare

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Posted 03 March 2010 - 01:39 AM

Hi :P

Do you cool your samples on ice or on a cooly block after denaturation? A few months ago, my labelling stopped working well and I realised my cooly block kinda broke down and wasn't cold enough! Perhaps it's a long shot. but thought it was worth mentioning :D

Clare



Hi all,

I'm doing indirect labeling (Aminoallyl + Cy3/5) for a couple of years. I used to get 250-500 pMol of each color with frequency of incorporation (FOI) 10+. Lately I started getting 60-100 pMol with FOI 2-6.

My prorocol is in brief:
1. Amplification with klenow, aminoallyl mix, and random primers ON.
2. The next day I clean with microcon
3. Coupling with the Cy dye in MaHCO3 0.1M buffer for 1.25h. I'm using a fresh buffer each time at pH 8.6.
4. Cleaning with Quiagen purification column.

I tried changing the Aminoallyl stock and the batch of the Cy dye, the klenow enzyme, the random primers. Does anyone have an idea what could be interrupting?

Thanks,
Idit



#3 Idit

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Posted 03 March 2010 - 04:55 AM

Hi Clare,
Yes, I cool the samples 5 minutes on ice. In what temperature do you do the coupling with the dye?
Thanks,
Idit

Hi :P

Do you cool your samples on ice or on a cooly block after denaturation? A few months ago, my labelling stopped working well and I realised my cooly block kinda broke down and wasn't cold enough! Perhaps it's a long shot. but thought it was worth mentioning :)

Clare



Hi all,

I'm doing indirect labeling (Aminoallyl + Cy3/5) for a couple of years. I used to get 250-500 pMol of each color with frequency of incorporation (FOI) 10+. Lately I started getting 60-100 pMol with FOI 2-6.

My prorocol is in brief:
1. Amplification with klenow, aminoallyl mix, and random primers ON.
2. The next day I clean with microcon
3. Coupling with the Cy dye in MaHCO3 0.1M buffer for 1.25h. I'm using a fresh buffer each time at pH 8.6.
4. Cleaning with Quiagen purification column.

I tried changing the Aminoallyl stock and the batch of the Cy dye, the klenow enzyme, the random primers. Does anyone have an idea what could be interrupting?

Thanks,
Idit



#4 Clare

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Posted 08 March 2010 - 03:02 AM

Hi again :)

I use a different method (Invitrogen BioPrime kit) but it's 37degC ON.
Clare

[quote name='Idit' date='Mar 3 2010, 12:55 PM' post='61059']
Hi Clare,
Yes, I cool the samples 5 minutes on ice. In what temperature do you do the coupling with the dye?
Thanks,
Idit




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