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cloning from PCR


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#1 dorightman

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Posted 24 June 2004 - 06:47 AM

i'm PCRing my gene (1.1kb) out from a plasmid template to create different restriction enzyme sites at the ends. I gel purify the PCR product using a kit from Qiagen and elute with 10mM TrisCl, pH8.0. I get a good recovery....i check this by running it out on a gel. from this estimated conc. i digest with BglII and XbaI and gel purify and test conc. the same as above. from this i set up a ligation in a vector:insert ratio of 3:1, 1:1, and 1:3 with ligase from Fisher. i transform all 10ul of ligation reaction into 100ul of competent cells and plate 100ul of the transformed cells, but still get no colonies.

what seems strange is after digesting, the vector (~3.2kb) shows a clear exised band of about 40bp, the fragment between BglII and XbaI, whereas my digested PCR product shows no small band, even when I ran a 20% acrylamide gel to look for extra small fragments (5bp and 8bp from each primers' 5' end). is this something i should be worried about? how can the enzyme sites NOT be there when I get a very dense band from my PCR reaction at 1.1kb?

#2 pcrman

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Posted 24 June 2004 - 12:04 PM

since you amplify your gene from a plasmid with primers into which restriction site is incorporated, one thing I can think of is how many extra bases outside the restriction site are in your primers.

Please check this two threads:

http://www.protocol-...hive/2/123.html
http://www.protocol-...ive/2/3053.html

#3 kant0008

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Posted 27 June 2004 - 04:46 PM

Hi,
I have very strong doubts you'd be able to see such small fragments no matter what you do. How far into your PCR products are your restriction sites? Enzymes normally need at least 6 bases distnace from recognition site to be present to cut. Also, do you have a transformation control (same plasmid but undigested, no insert) to make sure your cells are still competent?

#4 xysun

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Posted 29 June 2004 - 10:17 PM

why not ligate the PCR product into pGEM-T easy vector, and transform it into Ecoli, then pick the right plasmid to digest by your enzyme. this will be no problem, 2~3 days is ok.

#5 labrat

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Posted 30 June 2004 - 08:05 AM

I agree with xysun. Some time ago, after trying digesting PCR product and inserting into a vector without success, I gave up and did a TA cloning first and then cut the insert from the TA vector, which has guaranteed success.

#6 BioAdventurer

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Posted 18 July 2004 - 03:15 AM

I agree with those who say 40bp is toooo small to be checked on the gel. The only way to do that is to check your gel every 3-5' to see a band that will eventually faint after a while.

The problem that I had once was because my restriction sites, that I created using specific primers, where wrong. Instead of using the correct sequence in my EcoRI site, I made two base pairs wrong using the complementary sequence. This showed a product in my first elongation, but failed to show colonies, because simply didn't ligate to my vector.

Hope I've helped

#7 mab

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Posted 08 December 2004 - 08:25 AM

You can easily visualize 40 bp on a 4% agarose gel.

You should dilute your ligation mixture (ideally 5 times) before transformartion .... and

check for the competence of your cells (using a control- vector without insert)

#8 Daniel5306

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Posted 10 December 2004 - 09:44 AM

I am agreed with Mab, you need control when you did such kind of experiment. It really helps you when some thing wrong happens. Good luck!

Edited by Daniel5306, 10 December 2004 - 09:44 AM.


#9 syracuse_guy2000

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Posted 19 January 2005 - 02:18 PM

Sometimes the ligase could be a problem. Try Rapid Ligation Kit 2X buffer and ligate for overnight, it worked better for me than T4 ligase 10X buffer. Also make sure your insert:vector ratio is around 3:1.

#10 tuckern

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Posted 24 January 2005 - 01:45 AM

You could blunt end clone your fragment into pUC, then the digest should work. Remember that your vector should be de-phosphorylated and your insert phosphorylated.

#11 zhgljj1998

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Posted 31 January 2005 - 07:19 AM

i'm PCRing my gene (1.1kb) out from a plasmid template to create different restriction enzyme sites at the ends.  I gel purify the PCR product using a kit from Qiagen and elute with 10mM TrisCl, pH8.0.  I get a good recovery....i  check this by running it out on a gel. from this estimated conc. i digest with BglII and XbaI and gel purify and test conc. the same as above.  from this i set up a ligation in a vector:insert  ratio of 3:1, 1:1, and 1:3 with ligase from Fisher.  i transform all 10ul of ligation reaction into 100ul of competent cells and plate 100ul of the transformed cells, but still get no colonies.

what seems strange is after digesting, the vector (~3.2kb) shows a clear exised band of about 40bp, the fragment between BglII and XbaI, whereas my digested PCR product shows no small band, even when I ran a 20% acrylamide gel to look for extra small fragments (5bp and 8bp from each primers' 5' end).  is this something i should be worried about?  how can the enzyme sites NOT be there when I get a very dense band from my PCR reaction at 1.1kb?

man, how can you see the bank about 40 bp, 4%argrose gel?, i am going to check my double digested dna between BamH1 and xho1 (40bp) now, so need your suggestion, thank you.

#12 Freiberger

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Posted 23 February 2005 - 02:05 PM

Hi,
The RE Xba I is sensitive to dam methylation. Maybe a reason why you don't see any small fragment is because one of you RE didn't cut...

#13 sharath

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Posted 26 February 2005 - 06:29 AM

I believe there is some problem with your transformation procedure. Include a positive control to check the technique.

You are uisng 10ul of ligation mix with 100ul cells. that is 1:10 or 10%. That I beleive is way too high. try with 1ul and 5ul.
Sharath B.

#14 Scott

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Posted 04 March 2005 - 04:07 AM

From my experience 6 bases on the end is definately not enough with regards to digesting ends of PCR product for anything except maybe EcoR1. You could try blunting it in to your vector, or if you don't want the extra few bases blunt into a cloning vector and cut out of there to insert into your vector.

Cheers,

#15 Treffles

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Posted 16 November 2009 - 10:19 AM

Why not clone your pcr product directly into a TA vector (topo TA for example) before digesting or doing anything else? Just verify this vector for the presence of your restriction sites...




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