setting up a transformation--how will the competent cells be treated? 1. no plasmid (negative...
TRANSCRIPT
![Page 1: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/1.jpg)
Setting up a transformation--how will the competent cells be
treated?1. No plasmid (negative control, nothing
should grow on this plate)2. Supercoiled plasmid of a known
concentration (to determine efficiency of competent cells, in transformants/microgram)
3. Vector DNA (dephosphorylated?) ligated without insert DNA (background transformants)
4. Vector DNA ligated with insert DNA (desired products)
![Page 2: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/2.jpg)
Example outcome of a successful transformation: chemically competent
cells
1) No DNA--No colonies2) 2 nanograms (10-9 g, 10-3 micrograms)
supercoiled plasmid DNA--500 colonies (efficiency of cells: 2.5 x 105 transformants per microgram DNA)
3) Vector alone--small number of colonies4) Vector plus insert--larger number of
colonies than for #3
![Page 3: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/3.jpg)
Identifying recombinant plasmid-containing cells
• Alpha complementation: most white colonies represent presence of insert DNA blocking functional beta galactosidase
• Increase in number of transformants in presence of insert vs. absence of insert– Insert treated with alkaline phosphatase– Directional cloning--preventing religation of
vector– SCREEN colonies/plasmids for inserts, usually
by PCR
Confirm clones by sequencing
![Page 4: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/4.jpg)
Mobilizing DNA: vectors for propagation in E. coli
•Plasmids•Bacteriophage
M13 Lambda
•Specialized cloning vectors expression vectors and tags vectors for large pieces of DNA, e.g. Cosmids and BACs
![Page 5: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/5.jpg)
Bacteriophages: useful vectors in molecular cloning
I. Lambda: a “head and tail” phage
--The lambda life cycle--Basic cloning in lambda
II. M13: a filamentous phage--Life cycle--genome structure--phagemids
![Page 6: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/6.jpg)
Bacteriophages
• Viruses that infect bacteriaa) “head and tail”b) Filamentousc) etc….
• Nucleic acid molecule (usually DNA)– Carrying a variety of genes for phage replication– Surrounded by a protective protein coat (capsid)
• Infection (instead of transformation):– Phage attaches to outside of bacterium, injects DNA– Phage DNA is replicated– Capsid proteins synthesized, phage assembled and released
![Page 7: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/7.jpg)
Bacteriophage lambda
• “head and tail” phage, very well-studied• Large, linear genome--48.5 kb
– Central region of genome (“stuffer”) is dispensable for infectious growth--it can be engineered out
• Two lifestyle modes– Lytic: replicative mode– Lysogenic: latent mode
• Useful for cloning 5-25 kb DNA fragments
![Page 8: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/8.jpg)
lambda genome
![Page 9: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/9.jpg)
Lambda: lytic infection
decision
Linear DNA
![Page 10: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/10.jpg)
Lambda: latent infection (lysogeny)Lysogen: an E. coli strain that can be made to lyse under the right conditions (e.g. UV treatment)
![Page 11: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/11.jpg)
Lambda as a cloning vector
• Insertional vectors (clone into single restriction site, can only increase genome size by 5% (size of foreign DNA insert depends on the original size of the phage vector, about 5 to 11 kb)
• Replacement vectors (removing “stuffer”), can clone larger pieces of DNA, 8 to 24 kb (sufficient for many eukaryotic genes)
![Page 12: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/12.jpg)
Cloning in lambda phage--an overview
Left arm Right arm“Stuffer”
1) Restrict, purify right and left arms
2) Ligate with foreign DNA
3) “Package” ligation mixture into phage heads
4) Plate mixture on E. coli, individual plaques represent recombinant clones
![Page 13: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/13.jpg)
Examples of “replacement” lambda vectors
![Page 14: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/14.jpg)
The packaged
phage particles are
infectious
How to transfer recombinant
lambda into cells?
![Page 15: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/15.jpg)
Selecting recombinant lambda phages I
• There is a minimal size of DNA that can be packaged in lambda phage heads
• Remove stuffer (for some replacement vectors), the ligated “arms” cannot be packaged without an insert present
• Selection: only thing that is infectious is the recombinant DNA product
![Page 16: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/16.jpg)
Selecting recombinant lambda phages II
• Wild type lambda cannot grow on E. coli infected with phage P2 (spi, or sensitive to P2 inhibition), spi+ conferred by red and gam genes in “stuffer”
• Only phage lacking stuffer (they don’t have spi gene) can make plaques on lawn of E. coli containing a P2 lysogen
![Page 17: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/17.jpg)
Filamentous phages: M13
• Single-stranded, circular genome, 6.4 kb
• Can clone pieces of DNA up to 6X the M13 genome size (36 kb) -- but the larger the DNA, the less stable the clone is…..
• Useful for– Sequencing– Site-directed mutagenesis (later)– Any other technique that requires single stranded
DNA
• Drawback: foreign DNA can be unstable (slows down host cell growth, so deletions confer a selective advantage)
![Page 18: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/18.jpg)
M13 structure
Used in ‘phage display’ techniques
![Page 19: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/19.jpg)
M13 life cycle: an overview
ss
ss
dsIsolate for cloning
![Page 20: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/20.jpg)
M13: life cycleCell has to have F plasmid for infection to work
![Page 21: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/21.jpg)
M13:life cycle
Isolate phage (and single-stranded DNA) in supernatant
Isolate double-stranded DNA by standard plasmid prep
![Page 22: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/22.jpg)
M13 doesn’t lyse cells, but it does slow them down
M13 infections form plaques, but they are “turbid”
“lawn” of E. coli
![Page 23: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/23.jpg)
M13 mp18: engineered for alpha complementation
![Page 24: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/24.jpg)
Phagemids: plasmid/M13 hybrids• Plasmids containing both plasmid (colE1) origin and bacteriophage M13 origin of replication
•To recover single-stranded version of the plasmid (for sequencing, e.g.), infect transformed (male) strain with a helper phage (M13KO7)
• Helper phage cannot produce single stranded copies of itself, but provides replication machinery for single-stranded copies of the phagemid DNA
• Phagemid single stranded DNA is packaged and extruded into supernatant--can then be isolated for sequencing, etc.
![Page 25: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/25.jpg)
Uses of Bacteriophages:
Lambda -- large-ish DNA fragments•for gene cloning (large eukaryotic genes)•Excellent selection capability (stuffer stuff)•Clone lots of precisely-sized DNA fragments for library construction
M13 -- single-stranded DNA•Sequencing•Site-directed mutagenesis•Etc.
![Page 26: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/26.jpg)
Specialized vectors for E.coli
I. Expression vectors
II. Large DNA molecules: Cosmids, PACs, and BACs
Course packet: #25, 26, 27
![Page 27: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/27.jpg)
Expression vectors
• For production of specific RNA or protein of interest
• Optimized for transcription, translation, and post-translational handling
Typical expression vector cloning site:
promoter MCS
tags tags
Transcriptionterminator
![Page 28: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/28.jpg)
Expression vectors: RNA: expression occurs in vitro
(purified plasmids)
![Page 29: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/29.jpg)
Making micro RNAs for RNAi: one example
![Page 30: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/30.jpg)
![Page 31: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/31.jpg)
How to control transcription driving RNA/protein expression
in vivo?• T7 RNA polymerase promoters: T7 RNA
polymerase under control of lac repressor (induced by IPTG)
• Lambda PL promoter, controlled by lambda repressor (which is regulated by trp repressor)
• pBAD promoter, controlled by the araC protein in response to arabinose
![Page 32: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/32.jpg)
pET vectors: protein expression
![Page 33: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/33.jpg)
Helper tags for protein production and purification
• 6/7 histidine tag: interacts very specifically with Ni2+ ions, which can be immobilized on columns or beads
• Biotin carboxylase: covalently attaches to biotin, biotin binds to streptavidin which can be immobilized on columns or beads
• Epitopes (e.g. c-myc) for specific antibodies can be included as tags--purify on antibody column
• Tags can be engineered to be removable
![Page 34: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/34.jpg)
Using tags in protein purification
high affinity, high specificity
![Page 35: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/35.jpg)
A protein purification scheme--removable tag
![Page 36: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/36.jpg)
Cloning large DNA fragments• Cosmids: bacteriophage lambda-based• Bacteriophage P1 plasmids• BACs: F plasmid-based
replicon transfer
LambdacolE1
P1P1F
ARS
transfectiontransfectiontransfectionelectroporationelectroporationtransformation
This is a very good table to be familiar with
![Page 37: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/37.jpg)
Why clone large pieces of DNA???
Make libraries: genome broken up into small, manageable, organizable pieces
Each recombinant DNA fragment from the ligation--a piece of the genome
How many recombinant DNA molecules are required in a library to get complete coverage of a genome?
N = ln(1-p)
ln(1-f)
P = probability of getting a specific piece of the genome (1.0 = 100%)f = fractional size of clone DNA relative to genome
N = number of clones needed
![Page 38: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/38.jpg)
N = ln(1 - 0.99)
ln(1 - )1.7 x 104
3 x 109
99% probability of having a given DNA sequence
17 kb fragment library
Mammalian genome: 3 x 109 base pairs
N = 8.1 x 105 clones required
![Page 39: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/39.jpg)
Cosmids:
• 5 kb plasmids, antibiotic resistance, plasmid origin of replication
• Contain lambda cos sites required for packaging into lambda phage heads
• Packaging only occurs with 37-52 kb fragments--selection for large fragments
• Packaged DNA is inserted into cells and then replicates as a very large plasmid
![Page 40: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/40.jpg)
Cloning in a cosmid
Desired ligation Products--these are packaged
![Page 41: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/41.jpg)
Cloning in a cosmid
Instead of transformation, desired ligation products are packaged and then transfected into cells
Selection for colonies, not screening of plaques (not infectious)
![Page 42: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/42.jpg)
Cosmids: a specific cloning scheme
split
Sau3A: GATC 5’ overhang (compatible with BamHI sticky end)
Prevents ligation without insert
Prevents multiple fragments
![Page 43: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/43.jpg)
Phage P1 vectors: cloning up to 100 kb DNA fragments
85-100 kb
![Page 44: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/44.jpg)
Phage P1 vectors: cloning up to 100 kb DNA fragments
Efficiency of packaging is typically low: thus it is not good for making large genomic libraries
![Page 45: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/45.jpg)
Phage P1 vectors:cloning up to 100 kb DNA fragments
PACs: like P1 vectors but the DNA is not packaged (transfer by electroporation)
![Page 46: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/46.jpg)
BACs: Bacterial Artificial Chromosomes
• Based on the F factor of E. coli:--100 kb plasmid, propagates through conjugation--low copy number (1-2 copies per cell)--2 genes (parA and parB): accurate partitioning during cell division
• BACs: just have par genes, replication ori, cloning sites, selectable marker
• Can propagate very large pieces of DNA:•up to 300 kb
• Relatively easy to manipulate: move into cells by transformation (electroporation)
![Page 47: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/47.jpg)
General BAC vector
replication
selection
Cloning, etc
7 kb
![Page 48: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/48.jpg)
o---- Cloning strategies ----o
I. Making DNA “libraries” (from genomic DNA, mRNA “transcriptome”)
II. Screening to identify a specific clone (the needle in the haystack)-- by the sequence of the clone-- by the structure or function of the expressed product of the clone
Course reading: #28 (and 29)
![Page 49: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/49.jpg)
Overview of strategies for cloning genes
1)
2)
3)
4)
Get DNA
Ligate to vector
Transform or transfect
Look for the gene…
![Page 50: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/50.jpg)
Genomic DNA RNA
1) Get DNA
![Page 51: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/51.jpg)
Ligate to vector: how to make this reaction favorable?
![Page 52: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/52.jpg)
This yields a “library”, a representative set of all the pieces of DNA that make up a genome (or all the cDNAs that correspond to the “transcriptome”)
cDNAs from different tissues reflect the different RNA populations that you find in distinct cell types:Hence “liver” vs. “brain” vs. “heart” cDNA libraries
There are lots of ways to identify a particular gene…
![Page 53: Setting up a transformation--how will the competent cells be treated? 1. No plasmid (negative control, nothing should grow on this plate) 2. Supercoiled](https://reader036.vdocuments.net/reader036/viewer/2022081515/56649ce55503460f949b25ba/html5/thumbnails/53.jpg)
Overview of strategies for cloning genes