Sequencing Problems

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Gidget's picture
Sequencing Problems

Hi fellow lab rats,
I am having a problem with sequencing and wouldl like for some ideas as to where I can look for the issue.
So my protocol is:
1. PCR amplify  gene of interest, after purification and DNA concentration determination the products are run of a gel to check the quality.
2. The purified PCR product is then ligated into pDrive Vector
3. Transformed via heat shock into E. Coli (EZ competent cells)and then plated on a 1.5% agar plate with Carbonacillin.  After allowing the plate to grow overnight at 37C the plates were transferred to a 4C incubator. 
4. Clones were picked and grown in LB media + Car and allowed to grow at 37C overnichgt.
5. A mini prep was performed., DNA conc determined
DNA diulted 1:10 and sequenced.

No there is no alignemnt iwth DNA star for more than 85% of the clones picked!Andt solitions.
We performed a coloy PCR and all the clones had the right band for the palsmid
Any suggestions?

Biju's picture
Hi Gidget

Hi Gidget
I will sequence the pcr  product before cloning to ensure I amplified the correct sequnce needed.Hope this helps.

Gidget's picture
Thanks alot Biji!

Thanks alot Biji!
I have been doing this all along without having to sequence the PCR product and I've never encountered such a problem. Our primers and protocol have been going well.  Can you think of any other way this problem could've occurred. Regardless, your suggestion is great and barring other suggestions I'll have no choice but to do this but it adds to the complexity and time to my protocol.

The FFM's picture
 Are you sequencing with

 Are you sequencing with primers that start in the vector and sequence through the cloned fragment? 

have you tried using primers within your predicted cloned fragment and that will sequence out into the vector at either end to see in which orientation the fragment has cloned?

However before you blow a load of money doing lots more sequencing

Perform a PCR/ restriction digest on the mini preps - 

PCR approach

1 primer that anneals to the vector - 2nd primer predicted to anneal in the cloned fragment. - the size will tell you if the correct thing is in there and if it is in the correct orientation.

OR restriction digest

Perform a double digest using two unique cutters if you can find them.
1 that cuts the plasmid and the other that cuts the cloned fragment.  Again, the size of the fragments on the gel will tell you if you have cloned the right thing and in the right orientation.
The restrictions sites don't have to be unique - it just makes your life easier - if you cannot find a unique one in the cloned fragment, pick one that will give you something distinctive on the gel when it cuts and the fragment is inserted correctly.
Obviously preform the control double and single digests on the empty vector backbone too to make sure everything is cutting properly.

Maddy's picture
Hi, TheFFM

Hi, TheFFM

I too have the similar problem.

In my case, i get the fragment release of desired size after restriction digestion.

however the sequence does not match.
any suggestions