
cloning from PCR
#1
Posted 24 June 2004 - 06:47 AM
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
Posted 24 June 2004 - 12:04 PM
Please check this two threads:
http://www.protocol-...hive/2/123.html
http://www.protocol-...ive/2/3053.html
#3
Posted 27 June 2004 - 04:46 PM
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
Posted 29 June 2004 - 10:17 PM
#5
Posted 30 June 2004 - 08:05 AM
#6
Posted 18 July 2004 - 03:15 AM
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
Posted 08 December 2004 - 08:25 AM
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
Posted 10 December 2004 - 09:44 AM
Edited by Daniel5306, 10 December 2004 - 09:44 AM.
#9
Posted 19 January 2005 - 02:18 PM
#10
Posted 24 January 2005 - 01:45 AM
#11
Posted 31 January 2005 - 07:19 AM
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.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?
#12
Posted 23 February 2005 - 02:05 PM
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
Posted 26 February 2005 - 06:29 AM
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.
#14
Posted 04 March 2005 - 04:07 AM
Cheers,
#15
Posted 16 November 2009 - 10:19 AM