Note: this is a search engine friendly version of my lab notebook, please see the pdf version of this document for a more human friendly, printer friendly version.
THIS CHAPTER/PROJECT IS NOT ACTIVE
I still think the idea is useful; but it was taking up time that I didn't have. I need to graduate someday.
Hopefully this will be a short successful chapter. All I want to do is get a cloning proceedure that works without restriction enzymes. The TA-TOPO kits do this, but they aren't directional. The USER kit from NEB kinda does this, but it isn't general. It requires a 9-mer overhang thing that while cool, is a little long for what I want to do. Their 9-mer eliminates the need for ligase. The method I want to get going here will use ligase.
8.1 Motivation
I think the original motivation for USER cloning was that it was faster and didn't require the ligation step. I am more interested in a restriction enzyme free ligation procedure. There are a few situations where it would be nice to not have to cut your DNA with a restriction enzyme for example:
- you want to clone a gene, but one (or both) of the enzymes at for your insert sites have a site inside your insert. you either have to switch the insert sites on the vector or somehow mutate the site in your gene and hope it doesn't mess up the way the gene works.
- you are cloning random or randomized DNA where you can possibly know if you have the sites
- you are cloning large pieces of DNA where you almost certainly have one of the sites
- you want to circularize a big piece of DNA without cutting it or ligating on adaptors
I've encountered many of those problems already and the others, I see myself bumping into in the future as I try fancier ways to do DNA sequencing.
The basic idea behind USER cloning is shown in Figure . Notice that only that only 3' extensions are practical. When using restriction enzymes to cut the vector, the only ones that will be compatable (besides blunt, but if I wanted blunt cloning I wouldn't be screwing around with this protocol in the first place) are those that leave a 3' overhang and have a T at the last cutting position (see Figure for a list of potential enzymes). With USER cloning just replace this last T with a U and that provide the cut site for the USER enzyme mix. U in this case is uridine (the deoxyribose form of uracil). Most oligo synthesis companies will stick a U in for you for a small fee (e.g. IDT DNA).
Please see the pdf version for figures
Figure 8.1: Example of USER cloning using SalI, PacI, and USER primers.
An important point to consider with this approach is that most proof-reading Taqs are not compatable with primers that contain a U (it stalls the polymerase). The stratagene Pfu C
x is supposed to not have this problem. Otherwise, a non-proofreading taq will work, albeit with more errors.
- try cloning a gene using USER and a digested pNEB193 vector (does adding extra base pairs to the primers help with the cutting? test...)
- try cloning a gene using USER and a taq amplified pNEB193 vector with USER ends (i.e. completely restriction enzyme free)
8.3 Cloning araB
As an initial test of the USER cloning strategy, I'm going to try and clone araB from the genome into pNEB193. I choose pNEB193, because it has a SalI and a PacI site, so it is possible to try directional cloning. I'd prefer to have a vector where I could clone using some combination of AatII, PvuI, and SacI as they all leave 4-bp overhangs, but NEB doesn't have such a vector and I didn't feel like digging around forever to find such a vector. As it stands, I think having the U only 2-bp from the end creates a bigger challenge anyways, so if it works I'll be pretty confident that it'll work with the other 4-bp overhang cutters too.
I choose araB, because it's pretty long (1701 bp), and it's also the first gene I could think of on the top of my head. The length make it suitable to try some circularization ideas later on and still be able to visualize the results easily on agarose gels. AraB has a AgeI site inside of it that I can use to digest with after it is circularized.
> EG10053 araB (complement(70048..68348)) E. coli
atgGCGATTG CAATTGGCCT CGATTTTGGC AGTGATTCTG TGCGAGCTTT GGCGGTGGAC
TGCGCTACCG GTGAAGAGAT CGCCACCAGC GTAGAGTGGT ATCCCCGTTG GCAGAAAGGG
CAATTTTGTG ATGCCCCGAA TAACCAGTTC CGTCATCATC CGCGTGACTA CATTGAGTCA
ATGGAAGCGG CACTGAAAAC CGTGCTTGCA GAGCTTAGCG TCGAACAGCG CGCAGCTGTG
GTCGGGATTG GCGTTGACAG TACCGGCTCG ACGCCCGCAC CGATTGATGC CGACGGAAAC
GTGCTGGCGC TGCGCCCGGA GTTTGCCGAA AACCCGAACG CGATGTTCGT ATTGTGGAAA
GACCACACTG CGGTTGAAGA AGCGGAAGAG ATTACCCGTT TGTGCCACGC GCCGGGCAAC
GTTGACTACT CCCGCTACAT TGGTGGTATT TATTCCAGCG AATGGTTCTG GGCAAAAATC
CTGCATGTGA CTCGCCAGGA CAGCGCCGTG GCGCAATCTG CCGCATCGTG GATTGAGCTG
TGCGACTGGG TGCCAGCTCT GCTTTCCGGT ACCACCCGCC CGCAGGATAT TCGTCGCGGA
CGTTGCAGCG CCGGGCATAA ATCTCTGTGG CACGAAAGCT GGGGCGGCCT GCCGCCAGCC
AGTTTCTTTG ATGAGCTGGA CCCGATCCTC AATCGCCATT TGCCTTCCCC GCTGTTCACT
GACACTTGGA CTGCCGATAT TCCGGTGGGC ACCTTATGCC CGGAATGGGC GCAGCGTCTC
GGCCTGCCTG AAAGCGTGGT GATTTCCGGC GGCGCGTTTG ACTGCCATAT GGGCGCAGTT
GGCGCAGGCG CACAGCCTAA CGCACTGGTA AAAGTTATCG GTACTTCCAC CTGCGACATT
CTGATTGCCG ACAAACAGAG CGTTGGCGAG CGGGCAGTTA AAGGTATTTG CGGTCAGGTT
GATGGCAGCG TGGTGCCTGG ATTTATCGGT CTGGAAGCAG GCCAATCGGC GTTTGGTGAT
ATCTACGCCT GGTTTGGTCG CGTACTCGGC TGGCCGCTGG AACAGCTTGC CGCCCAGCAT
CCGGAACTGA AAACGCAAAT CAACGCCAGC CAGAAACAAC TGCTTCCGGC GCTGACCGAA
GCATGGGCCA AAAATCCGTC TCTGGATCAC CTGCCGGTGG TGCTCGACTG GTTTAACGGC
CGCCGCACAC CGAACGCTAA CCAACGCCTG AAAGGGGTGA TTACCGATCT TAACCTCGCT
ACCGACGCTC CGCTGCTGTT CGGCGGTTTG ATTGCTGCCA CCGCCTTTGG CGCACGCGCA
ATCATGGAGT GCTTTACCGA TCAGGGGATC GCCGTTAATA ACGTGATGGC ACTGGGCGGC
ATCGCGCGGA AAAACCAGGT CATTATGCAG GCCTGCTGCG ACGTGCTGAA TCGCCCGCTG
CAAATTGTTG CCTCTGACCA GTGCTGTGCG CTCGGTGCGG CGATTTTTGC TGCCGTCGCC
GCGAAAGTGC ACGCAGACAT CCCATCAGCT CAGCAAAAAA TGGCCAGTGC GGTAGAGAAA
ACCCTGCAAC CGTGCAGCGA GCAGGCACAA CGCTTTGAAC AGCTTTATCG CCGCTATCAG
CAATGGGCGA TGAGCGCCGA ACAACACTAT CTTCCAACTT CCGCCCCGGC ACAGGCTGCC
CAGGCCGTTG CGACTCTATA A
Forward
ATG GCG ATT GCA ATT GGC CTC G
MT: 61.1C
Forward + Adaptor
AGCU ATG GCG ATT GCA ATT GGC CTC G
MT: 63.6C
Reverse
TTA TAG AGT CGC AAC GGC CTG GG
MT: 60.9C
Reverse + Adaptor
AU TTA TAG AGT CGC AAC GGC CTG GG
MT: 60.9C
8.3.1 PCR with uradine primers, Linearization of pNEB193
Wed Nov 1 15:27:23 EST 2006
PCR with uradine primers
Wed Nov 1 15:27:27 EST 2006
The uridine araC primers were reconstituted to 100 mM. A primer mix at 10 mM was made with the forward and reverse primers (all primer stocks were made with TE).
The PCR was performed according to the Stratagene Cx manual which recommended an annealing temperature 5C below the lowest MT of the two primers (this ended up being 55C). The rxn was: 500 nM each primer, 1
ml dNTP (200 uM final), 1
ml PfuCx polymerase (2.5U), 40
ml H
2O , and appx 100 ng genomic DNA.
This is a true hot-start polymerase. Hot-start was 2 min at 95C. Extension was 1 minute. Polish was 10 min at 72C. I ran 30 cycles. 10
ml of the PCR product was run on an 1%
ml agarose gel (see Figure ).
Please see the pdf version for figures
Figure 8.2: Digested pNEB193 and araB PCR with uridine
Brief Conclusions: The PCR definitely didn't yield the correct band of 1700 bp (Figure
8.2A). I'm not sure what those bands are? I'm going to try the PCR with a non-proofreading Taq.
Linearization of pNEB193
Nov 1, 2006
pNEB193 was ordered from NEB. I haven't made a freezer stock yet, so I'll just use their plasmid until I clone it and make my own.
I linearized the plasmid with blunt cutter SmaI. This will reduce any background transformations because the blunt ends shouldn't religate quickly. I also wanted to look at the plasmid one a gel one time before I used it.
The digestion was: 4
ml of pNEB193 (2
mg ), 1
ml NEBuffer4, 4.5
ml H
2O , 0.5
ml SmaI. Digested for 10 minutes at RT. Heat deactivated 20 minutes at 65C. I ran 2.5
ml in each of the pNEB193 lanes in Figure
8.2A.
Brief Conclusions: Linearization worked well, I don't see any other bands besides the linearize plasmid in Figure
8.2.
PCR with uradine primers and non error-correcting Taq
Nov 2, 2006
I want to determine if it is the PfuCx taq that is messing things up with the Uradine containing araB primers. I ran the following reactions:
Taq rxn: 0.5 genomic (125 ng), 2.0
ml primer mix, 25
ml NEB Taq master mix, 22.5
ml H
2O
PfuCx rxn: 0.5 genomic (125 ng), 2.0
ml primer mix, 1
ml dNTP, 5
ml PfuCx buffer, 40.5
ml H
2O
I ran 10
ml of each on a 1% agarose gel (see Figure
8.2B).
Brief Conclusions: The Taq PCR definitely got the correct band of 1700 bp (Figure
8.2B), but it also got a lot of other junk. The PfuCx got closer to the right band size (though it might be 100 bp short?), but it also got even more junk than the normal Taq. I think my strategy for now is to order new primers for different genes (I'll return to araB later depending on how the new test works). I want to try lrp (from genomic) and mCherry protein (from plasmid). I'm going to order two sets of primers for each gene: 1 with U's and the other with T's. Hopefully this will clear up what's going on. I'm going to order these from invitrogen which allows a smaller yield and it quite a bit cheaper than IDT for these Uridine based oligos. I also want to get the primers with a higher melting temperature than last time (which was 60C, bump it up to 65C this time).
8.4 Trying with mCherry and lrp
Nov 7, 2006
Didn't have much luck USER cloning araB. I want to see if it a problem with IDT, araB, melting temperature, PfuCx, or if it just isn't going to work. I'm going to try one plasmid based gene (should be easier to amplify) and one genomic gene.
8.4.1 mCherry and lrp USER primer design
mCherry forward
ATG GTG AGC AAG GGC GAG GAG GAT AAC ATG GCC
33bp 68C
mCherry forward + adaptor
AGCU ATG GTG AGC AAG GGC GAG GAG GAT AAC ATG GCC
37bp 71C
mCherry reverse
TTA CTT GTA CAG CTC GTC CAT GCC GCC GGT GGA G
34bp 70C
mCherry reverse + adaptor
AU TTA CTT GTA CAG CTC GTC CAT GCC GCC GGT GGA G
36bp 70C
lrp forward
ATG GTA GAT AGC AAG AAG CGC CCT GGC AAA GAT C
34bp 68C
lrp forward + adaptor
AGCU ATG GTA GAT AGC AAG AAG CGC CCT GGC AAA GAT C
38bp 70C
lrp reverse
TTA GCG CGT CTT AAT AAC CAG ACG ATT ACT CTG CTT GA
38bp 68C
lrp reverse + adaptor
AU TTA GCG CGT CTT AAT AAC CAG ACG ATT ACT CTG CTT GA
40bp 68C
This time I ordered the primers from invitrogen. They synthesize the lower amounts (25nM) for U containing primers, so it works out much cheaper ($12 a primer for these long ones with U, 5 dollars for the ones with a T). I've seen other people use the ones from Invitrogen, so hopefully these will work better than the ones form IDT.
8.4.2 USER PCR of mCherry and lrp
Resuspend primers at 100
mM . Then make a mix of 10
mM . Use 2
ml per 50
ml rxn for 400 nM final conc. I diluted the mCherry miniprep from section page to be 40 ng/
ml .
Taq rxn: 0.5 genomic (125 ng), 2.0
ml primer mix, 25
ml NEB Taq master mix, 22.5
ml H
2O
PfuCx rxn: 0.5 genomic (125 ng), 2.0
ml primer mix, 1
ml dNTP, 5
ml PfuCx buffer, 40.5
ml H
2O
Taq cherry rxn: 0.5 plasmid (20 ng), 2.0
ml primer mix, 25
ml NEB Taq master mix, 22.5
ml H
2O
PfuCx cherry rxn: 0.5 plasmid (20 ng), 2.0
ml primer mix, 1
ml dNTP, 5
ml PfuCx buffer, 40.5
ml H
2O
Thu Nov 9 13:03:09 EST 2006
Actually, I just opened the envelope and noticed I only recieved the nonUSER primers. I guess it'll take them a little longer to make the ones with the U's. Save this exp for another day....
Mon Nov 13 19:06:52 EST 2006
Got the primers, running the PCR rxns overnight.
Wed Nov 15 14:40:23 EST 2006
I ran the PCR rxns on a 1.5% agarose gel.
Please see the pdf version for figures
Figure 8.3: lrp and mCherry PCR with and without uridine using PfuCx and standard Taq. Gel is 1.5% run for 50 minutes at 100V.
I quantified the PCR yields from the gel using the using the Versadoc software:
| Linear extrapolation (per ml ) | Point-to-point extrapolation (per ml ) |
| TCU | 223 ng (28 ng/ml ) | 93 (12 ng/ml ) |
| PCU | 382 ng (48 ng/ml ) | 143 (18 ng/ml ) |
| TLU | 171 ng (21 ng/ml ) | 77 (10 ng/ml ) |
| PLU | 351 ng (44 ng/ml ) | 133 (17 ng/ml ) |
| TCN | 218 ng (27 ng/ml ) | 91 (11 ng/ml ) |
| PCN | 386 ng (48 ng/ml ) | 144 (18 ng/ml ) |
| TLN | 163 ng (20 ng/ml ) | 74 (9 ng/ml ) |
| PLN | 269 ng (34 ng/ml ) | 107 (13 ng/ml ) |
My gut feeling is that these are underestimates. I'll use the linear extrapolations for the USER cloning since these are always higher.
Brief Conclusions:
Wed Nov 15 15:51:16 EST 2006
It looks like everything is working just right with these Invitrogen primers. I don't know if it is the company, the increased length/mt, or the gene. I can now trying to clone these guys in using the USER enzyme. If the cloning step works, maybe I'll try to take one stab at figuring out why the araB didn't work (i.e. try invitrogen same primers and longer primers).
8.4.3 USER cloning of mCherry and lrp
Here we go...
preparing the pNEB193 vector
Thu Nov 16, 2006
I linearized the pNEB193 vector using SmaI in the following reaction: 4
ml pNEB193 (2
mg ), 0.5 SmaI, 1
ml Buffer4, 4.5
ml H
2O . I incubated at RT for 30 minutes and deactivated at 65C for 20 minutes.
This blunt-linearization was to prevent false positives in the ligation and to make the sticky ended inserts so short that they'd be easy to purify with a PCR purification (Qiagen).
After linearization, I added 1
ml Buffer1, 2
ml BSA, 6
ml H
2O , 0.5
ml PacI, and 0.5
ml SacI and incubated at 37C for 45 minutes followed by at 20 minute deactivation at 65C. The vector was cleaned up with a Qiagen PCR purification kit and eluted into 30
ml of EB buffer.
| Sample | DNA (ng/ul) | 260/280 | 260/230 | total yield |
| cut pNEB193 | 46.0 |
|
| 1.26 mg |
USER, ligation, transformation
Thu Nov 16, 2006
I pretty much just followed the instructions from the USER manual on the NEB website. I used their suggestion of adding ligase however, because my overhangs are much smaller than the ones used for the product that they sell.
20 ng vector, 10
ml PCR, 1
ml USER enzyme: incubate 15 minutes at 37 C.
add 1
ml T4 DNA ligase buffer, add 1
ml T4 DNA ligase: incubate at RT for 15 minutes.
I transformed 2
ml of each ligation/USER reaction into DH5
a. I plated 50
ml . I ran 5 total reactions: (using the nomenclature from the previous section) PUC, PUL, PNC, PNL, - control (plasmid only to make sure the blunt digestion and Qiagen cleanup efficiently removed the insert). The plates contained x-gal and IPTG for blue/white screening.
Brief Conclusions: how many colonies, did it work? are they white? if so pick for miniprep, pcr check and sequencing
Insert check of USER clones
I picked 8 clones, grew them overnight, miniprepped them, and checked the inserts by PCR using the M13 primers. The mCherry clone overnights were bright red, indicating that the correct gene was there.
Nov 29, 2006
I ran a PCR on the minipreps and ran 10
ml of the PCR rxn onto an agarose gel (Figure ).
Please see the pdf version for figures
Figure 8.4: Insert check for lrp and mCherry clones with and without uridine using PfuCx and standard Taq. Gel is 1.0% run for 50 minutes at 100V.
Brief Conclusions: The colored proteins show that the right thing is in there, even for the genes were I didn't have sticky ends. None of the inserts really amplified (Figure
8.4). All you can really see are the plasmids and the primers. I'm not sure what's going on.