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Help cloning!!!


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#1 micky74

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Posted 08 May 2009 - 04:52 AM

Hello every body

I hope someone can gimme a clue!

Problem: I need to clone a fragment of PCR of about 4.2 Kb in a plasmid of about 5.3Kb. Oligos are containing 2 different ER sites: BamH1 and Xho1. BamH1 sites is 4 nucleotide away from the end (5 if you count the A), XHo is 5 or 6 far away from the end. Xho and Bam are 50 bp far away in the vector.
Protocol

I digest 4 ug of vector and all the PCR product with bamh1 for 6 hrs. Purify with the qiagen purification kit. Then XhoI overnight. All enzymes with the appropriate buffer. Then heat inactivate enzyme at 65 C for 20 mins. Then treat vector with SAP (2U for 60 mins). Gel purify PCR and vector. I can see a single band for the vector and the band for the PCR. I ran also a control for the 2 digestion for the vector for the digestion with BamH1 and XhoI and I can see they worked fine. Ok gel purify then the PCRs and the vector with Qiagen kit. I got my PCR product from a nested to have a lot of product. So after gel extraction I read concentration at nanodrop. 15 ng/ul for the plasmid and 80 ng /ul for the PCR (more or less they have same lenght and you can see). So I tried 3 different reactions to do not waste many cells. Insert: vector ratio 3:1 (with 100 ng vector), then vector treated with SAP with no insert and then I just wanted to be sure to have some colonies and since I have had problems in the past with SAP treatment I set up a 3:1 with a SAP untreated aliquot of the plasmid. Basically I have few colonies in the SAP treated with no insert, no colonies at all in vector + insert and more colonies in vector SAP untreated + insert, but I haven't set up a control for this.

Last week I have done the same protocol starting with much more plasmid (8 ug) and not treated the vector at all with SAP. I plated the 1:1 insert:vector, 3:1 and vector alone. In that case I had lot of colonies in the vector alone, 2-3X more in the 1:1 ratio and 5-10 less in the 1:3 ratio. until now no positive colonies. I used to run a colony PCR with a primer on the insert and a primer on the vector. For lot of colonies I get a band (even enough strong), but when I replat thm they are negative. This makes me think I shoud have the product at least, but I can not find a single colony bearing the clone!!!

Anyone can help!

#2 phage434

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Posted 08 May 2009 - 05:25 PM

BamHI cannot be heat inactivated, but that is unlikely to be the problem. Why are you not simply digesting with both enzymes at the same time? I would be careful about UV exposure of your gels trashing your DNA. Make sure you are purifying your DNA to eliminate pcr enzymes before cutting with REs. Otherwise, you will fill in or cut back the critical overhangs produced by cutting.

I would:
PCR
column cleanup
cut both vector and PCR product with BamHI and XhoI in buffer 4 + bsa
SAP treat the vector (or not). You can probably do this directly in the RE buffer, possibly with added salts.
column clean up both cut vector and cut PCR product
ligate
transform

#3 micky74

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Posted 09 May 2009 - 02:26 AM

BamHI cannot be heat inactivated, but that is unlikely to be the problem. Why are you not simply digesting with both enzymes at the same time? I would be careful about UV exposure of your gels trashing your DNA. Make sure you are purifying your DNA to eliminate pcr enzymes before cutting with REs. Otherwise, you will fill in or cut back the critical overhangs produced by cutting.

I would:
PCR
column cleanup
cut both vector and PCR product with BamHI and XhoI in buffer 4 + bsa
SAP treat the vector (or not). You can probably do this directly in the RE buffer, possibly with added salts.
column clean up both cut vector and cut PCR product
ligate
transform



Hi
Thanks for the answer
The protocol I'm following is more or less the same. I perform 2 digestion since the enzymes are left over from other people and they are from different brand.
The digestions for the plasmid at least work fine independently since I always check with control plasmid they have worked.
I do column clean up after PCR- digestion BamhI-column clean up-digestion with XhoI- heat inactivation (that works for Xho I) gel extraction- ligation
For plasmid is more or less the same I do not perform column clean up after miniprep but the control is digested fine so I do not think is the problem.

I do think the major problem is gel extraction. I perform it carefully, but the fact that adding more PCR product the ligation doesn't work let me think I'm adding some inhibitor....but that is my opinion now...so I have skipped now the gel extraction and doing only column purification following the protocol you wrote.

Just few question for everyone.

Ligase reaction is performed in 20 ul. I usually do the reaction at 4 C for 16h. Ligase is T4 from Fermentas using 1-1.5 U. Anyone has found a volume or temperature that works better? How much ng of plasmid are you using? I 'm using 100 ng of plasmid but maybe is too much. 3:1 insert:vector is usually working?
Using the TOP10 cells from invitrogen should I follow the protocol from them? 30 mins ice- heat shock for 30' at 42 and 1hr soc- then plate?

Thanks for your suggestion again!

#4 kajmak

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Posted 09 May 2009 - 02:55 AM

As phage said digest with both enzymes at the same time, even if they are from different companies, they might work well together in a buffer from third company. You don’t need SAP and certainly not heat inactivation if you run it on a gel.

Anyway, if I were you I would do ligation in 20 um, add buffer, 1-2 um of your ligase and the rest divide between fragments. Eg. after 2 um of 10 X buff and 2 um of ligase, you need 8 um of vector and 8 um of insert – don’t even bother with ng. Ligate at room temperature for 2-3 hr (or 4 C overnight if you are patient).

The only other thing I would change in your method is heat shock during transformation. Increase it from 30 sec to 60-90 sec.

#5 micky74

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Posted 09 May 2009 - 03:22 AM

As phage said digest with both enzymes at the same time, even if they are from different companies, they might work well together in a buffer from third company. You don’t need SAP and certainly not heat inactivation if you run it on a gel.

Anyway, if I were you I would do ligation in 20 um, add buffer, 1-2 um of your ligase and the rest divide between fragments. Eg. after 2 um of 10 X buff and 2 um of ligase, you need 8 um of vector and 8 um of insert – don’t even bother with ng. Ligate at room temperature for 2-3 hr (or 4 C overnight if you are patient).

The only other thing I would change in your method is heat shock during transformation. Increase it from 30 sec to 60-90 sec.



when you say um you mean ul? I guess maybe 8ul of vector is too much especially if I need to follow the 3:1 or 5:1 insert:vector ratio. I checked on gel and the band of vector is quite brilliant whilst the PCR is 2-3X less, so maybe I need to add more PCR product

Is the time for heat shock going to change anything?

Cheers again

#6 kajmak

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Posted 09 May 2009 - 03:17 PM

I did mean uL, sorry. Try to add more PCR.

I just remembered, recently my ligation involving BamHI and XhoI sites (I had 3 fragments) didn’t work. My BamHI and XhoI sites came together, so BamHI/XhoI fragment wasn’t in a final vector.
It shouldn’t happen, but…

SacI forms complementary ends with XhoI, it can be added to PCR instead of XhoI. Due to 5’ and 3’ overhangs there is no way Bam and Sac will ligate.

Increased time of heat shock will make more pores in cells, but if you are getting colonies I would not worry about that.

#7 micky74

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Posted 11 May 2009 - 12:06 AM

I did mean uL, sorry. Try to add more PCR.

I just remembered, recently my ligation involving BamHI and XhoI sites (I had 3 fragments) didn’t work. My BamHI and XhoI sites came together, so BamHI/XhoI fragment wasn’t in a final vector.
It shouldn’t happen, but…

SacI forms complementary ends with XhoI, it can be added to PCR instead of XhoI. Due to 5’ and 3’ overhangs there is no way Bam and Sac will ligate.

Increased time of heat shock will make more pores in cells, but if you are getting colonies I would not worry about that.



Topic solved I guess. 0 colonies on vector alone treated with SAP, 60-80 clonies on 3:1, 5:1 insert:vector ratio. I guess my main problem was gel purification that I skipped in this experiment. Can not say if it was UV exposure or something in Qiagen kit inhibithing my reaction

I used the protocol I was using before

PCR
column cleanup
digestion
column purification
digestion
SAP treat the vector (or not). You can probably do this directly in the RE buffer, possibly with added salts.
column clean up both cut vector and cut PCR product
ligate
transform

Cheers again for the help

Edited by micky74, 11 May 2009 - 12:07 AM.


#8 micky74

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Posted 12 May 2009 - 09:27 AM

I did mean uL, sorry. Try to add more PCR.

I just remembered, recently my ligation involving BamHI and XhoI sites (I had 3 fragments) didn’t work. My BamHI and XhoI sites came together, so BamHI/XhoI fragment wasn’t in a final vector.
It shouldn’t happen, but…

SacI forms complementary ends with XhoI, it can be added to PCR instead of XhoI. Due to 5’ and 3’ overhangs there is no way Bam and Sac will ligate.

Increased time of heat shock will make more pores in cells, but if you are getting colonies I would not worry about that.



Topic solved I guess. 0 colonies on vector alone treated with SAP, 60-80 clonies on 3:1, 5:1 insert:vector ratio. I guess my main problem was gel purification that I skipped in this experiment. Can not say if it was UV exposure or something in Qiagen kit inhibithing my reaction

I used the protocol I was using before

PCR
column cleanup
digestion
column purification
digestion
SAP treat the vector (or not). You can probably do this directly in the RE buffer, possibly with added salts.
column clean up both cut vector and cut PCR product
ligate
transform

Cheers again for the help



I was too optimistic.

So I screened like 150 colonies and no positive results. I guess the vector is binding something I can not see but should be there on the gel and should be smaller, so I'm back to my problem and I need to purify my band of interest by gel extraction.
Any suggestion to perform it at the best? How much plasmid do you use for the ligation? I'm following fermentas protocol which gives 20 ul final volume and 50-400 ng of plasmid but actually I do not think I have that mcu PCR product to go to 5:1 insert: vector ratio

So any suggestion?

Cheers

#9 phage434

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Posted 12 May 2009 - 04:56 PM

So, what ARE your transformants if they are not what you want. Their identity will be a clue to what is wrong.

#10 micky74

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Posted 12 May 2009 - 11:42 PM

So, what ARE your transformants if they are not what you want. Their identity will be a clue to what is wrong.



Should I sequence them then?

#11 phage434

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Posted 13 May 2009 - 04:19 AM

I would prep and at least RE cut four to see what bands were present. If you have cheap easy sequencing, that would be best. You're working too hard if you are analyzing 150 colonies (at least without robotics to help).

#12 micky74

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Posted 13 May 2009 - 04:46 AM

I would prep and at least RE cut four to see what bands were present. If you have cheap easy sequencing, that would be best. You're working too hard if you are analyzing 150 colonies (at least without robotics to help).



Thanks phage

I analyzed three clones and if they look bigger than the original plasmid I analyzed them by fast digestion with BamHI and XhoI, so I can see only the linear vector, I can't see the insert, so I do think there is something smaller that enter the plasmid or double digestion is not working!!




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