The following is a description of the steps followed in identifying and screening microsatellite molecular markers for the Galápagos marine iguana (Amblyrhynchus cristatus).

Preparation of Partial Genomic Library
    Preparation of DNA Fragments and Plasmid/Vector for Ligation
    Ligation of Plasmid/Vector and DNA Fragments
    Preparation of Cells for Insertion of DNA Fragments
    Transformation of Compentent Cells
    Plating Out Transformed Cells
Screening of Partial Genomic Library
    Screening Colonies for Potentially Useful Microsatellite Sequences
        Colony Growth
        Oligonucleotide Labeling
        Hybridization and Detection of DIG Labeled Probes
        Selection of Colonies for Plasmid Extraction
Secondary Screening of Plasmids and DNA Inserts
Selection of Plasmids to Send Out for Sequencing
Design and Testing of Primers
Solution Recipes

A general overview of the process in graphical form is available and will pop-up as a new window that may be moved around and referenced as needed.  (An even more simplified version is also available.)  Specific procedures are outlined below.
 

Preparation of DNA Fragments and Plasmid/Vector for Ligation:

DNA that had been extracted by phenol-chloroform was restricted (cut) using Mbo 1 (which cuts at the same position as Sau 3A1). [Abril 2]

200 µl DNA (100-300 ng/µl)
25 µl 10x Buffer K
5 µl Mbo 1
adjust volume to 250 µl with PCR grade water

Digest 3 hours at 37^oC (with occasional mixing/tapping)

Inactivated enzyme by incubating at 80^oC for 15 minutes

Plasmid (pUC19 / map) was cut with Bam H1.

2 µl pUC19 (measured by UV 260/280 at 200 ng/µl)
2 µl 10x Buffer K
1 µl Bam H1
15 µl PCR grade water

Digested 1 hour 30 minutes at 30^oC

Inactiveated enzyme by incubating at 80^oC for 15 minutes

Separated out and saved 10 µl to use as a ligation control later on

Plasmid (pUC19) was dephosphorylated using Shrimp Alkaline Phosphatase (SAP).

10 µl restricted pUC19
1.2 µl 10x buffer
1 µl SAP

Incubated 10 minutes at 37^oC

Inactiveated enzyme by incubating at 65^oC for 15 minutes

Restricted pUC19 samples were extracted using phenol/chloroform/isoamylalcohol.

12 µl pUC19 sample
200 µl TE
200 µl phenol/chloroform/isoamylalcohol (25:24:1)

Vortexed (shook VERY well) 10 seconds and then centrifuged at 21,000g for 1 minute
Removed top aqueous layer (containing DNA) to a new microfuge tube
Added 20 µl 4M LiCl and mixed
Added 500 µl 100% ethanol (non-denatured) and inverted several times
Put into -20^oC overnight
Centrifuged at 21,000g for 1 minute
Poured off ethanol and allowed pellet of DNA to dry
Resuspended plasmid in TE and quantified by UV (260/280)
Diluted plasmid DNA concentration with TE to 100 ng/µl

Restricted DNA was extracted using phenol/chloroform/isoamylalcohol.

250 µl restricted DNA sample
200 µl TE
200 µl phenol/chloroform/isoamylalcohol (25:24:1)

Vortexed (shook VERY well) 10 seconds and then centrifuged at 21,000g for 1 minute
Removed top aqueous layer (containing DNA) to a new microfuge tube
Added 40 µl 4M LiCl and mixed
Added 1 ml 100% ethanol (non-denatured) and inverted several times
Put into -20^oC for a few hours to precipitate DNA
Centrifuged at 21,000g for 4 minutes
Poured off ethanol, added 1 ml 70% ethanol, and inverted several times
Centrifuged at 21,000g for 4 minutes
Poured off ethanol and allowed pellet of DNA to dry
Resuspended DNA in 20 µl TE and quantified by UV (260/280)

Checked restriction and extraction products on a 4% polyacrylamide gel.
 

Ligation of Plasmid/Vector and DNA Fragments (inserting fragments into circular DNA):
[Marzo 31]

1 µl cut pUC19 plasmid DNA (measured at 200 ng/µl)
2 µl digested DNA (100 ng/µl, 2x pUC19 to match DNA amounts)
1 µl T4 DNA ligase
1 µl 10x T4 DNA ligase buffer
5 µl PCR grade water

Incubated at 16^oC overnight

Preparation of Cells (E. coli) for Insertion of DNA Fragments:
(ensuring sterile technique at each step) [Marzo 13/14]

Prepared overnight cultures for inoculation of growth media.

Sterile toothpick was used to remove 2-5 colonies of E. coli from a petri dish (previously spread and grown overnight followed by storage at 4^oC)
Toothpick was placed into sterile test tube containing 4 ml LB broth
Tubes containing toothpicks were incubated at 37^oC overnight shaking at 200 RPM

Preparation of competent cells from overnight cultures (so plasmid DNA can be inserted).

Flasks containing 50 ml LB broth were inoculated with 0.5 ml fresh overnight culture
Incubated flasks at 37^oC shaking at 190 RPM until spectrophotometer A₆₀₀ (absorbance at a wavelength of 600 nm) reading was between 0.25-0.4
Transferred cell culture to 50 ml centrifuge tubes and cooled cells for 5 minutes on ice
Centrifuged at 3500 RPM for 10 minutes at 4^oC (Beckman benchtop model)
Poured off liquid and gently resuspended cells in 20 ml sterile ice-cold 5 M CaCl₂
Incubated on ice for 30 minutes
Centrifuged at 3500 RPM for 10 minutes at 4^oC
Poured off liquid and gently resuspended in 4 ml sterile ice-cold CaCl₂

Transformation of Compentent Cells (inserting the DNA): [Abril 6]

Diluted ligation results by mixing 5 µl ligation solution with 10 µl Tris-HCl buffer
Put 200 µl competent cells and 12 µl diluted ligation result (from above) into a sterile glass tube (lesser quanity - 5 µl ligated solution - recommended in protocol did not produce good results)
Incubated on ice for 45 minutes
Incubated in a water bath at 42^oC for 3 minutes (with NO shaking)
Chilled in an ice bath for 1 minute
Removed from the ice bath and added 800 µl of 37^oC LB broth
Incubated at 37^oC for 60 minutes with shaking every 10-15 minutes

Plating Out Transformed Cells (detection of cells with inserted plasmids): [Abril 7]

Prepared LB/ampicillin/X-gal/IPTG plates [Marzo 31]

Poured LB agar/ampicillin plates (10 cm) containing 1 ml/L ampicillin solution (added after autoclaved LB agar had cooled to "touchable" temperature) and cooled
Mixed a 1:1 solution of X-gal and IPTG solutions
Spread 50 µl of 1:1 solution evenly on the surface of the cool plates
Allowed to soak in at 37^oC for 4 hours

Spread 200 µl of transformed cells over each plate
Allowed spread cell solution to soak in before turning plates over
Incubated at 37^oC until colonies were distinct and blue/white detection system was visible

An overview of the screening procedure is available as a pop-up window for reference.  The specific steps and procedures are outlined below:
 

Screening Colonies for Potentially Useful Microsatellite Sequences:

Colony Growth
Poured LB agar/ampicillin plates (10 cm x 10 cm), but no IPTG or X-gal (see above)
Using sterile toothpicks patched over (touching toothpick to original bacterial colony, to a labeled plate, and then again to a replicate plate) colonies that had plasmids (those that were white rather than blue) onto 26x26 gridded plates
Incubated at 37^oC until there was substantial bacterial growth
Transfered to 4^oC for storage [Abril 10/11]

Oligonucleotide Labeling
Had oligonucleotides synthesized by Genosys based upon the recommendations found in the document "PROTOCOLES MICROSATELLITES / MICROSATELLITE PROTOCOLS" - written by Arnaud Estoup and Julie Turgeon, 12/1996

 

(TC)10	(TG)10	(CAC)5CA
CT(CCT)5	(TGTA)6TG	CT(ATCT)6

Rehydrated oligonucleotides containing repeat sequences in TE to 100 pmol/µl
Labeled oligos according to the protocol provided with the DIG Oligonucleotide 3'-End Labeling Kit purchased from Roche Molecular Biochemicals [Abril 17/18]

Hybridization and Detection of DIG Labeled Probes
Cut positively charged nylon membranes to fit petri dishes containing grids of bacterial colonies [Abril 24/25]
Labeled membranes with a ballpoint pen along the top edge in multiple places
Carefully placed membranes on surface of petri dishes for a few minutes (being sure not to drag at any time)
Removed membrane and allowed to air dry (usually 10-30 minutes)
Treated each membrane by the following four soaking (NOT submerging) steps:

10% SDS - 3 minutes
Denaturing solution - 5 minutes
Neutralizing solution - 5 minutes
2x SSC - 5 minutes

Let membrane air dry (approximately 30 minutes)
Crosslinked (bound) DNA to membrane in a UV Stratalinker on the "automatic" setting (120,000 µJoules)
Pre-heated hybridization oven, rollers, and PerfectHyb Plus (from Sigma) hybridization buffer to 46^oC
Threaded a string through that top labeled portion of the membranes (to hold them in place so they wouldn't slide around - with the string pinched between the cover and glass of the roller)
Placed them in the warmed rollers containing warmed buffer and pre-hybridized for 10-15 minutes
Added 2 µl of each labeled oligo (12 µl total) to each hybridization tube
Hybridization at 46^oC continued overnight
Washed with low stringency wash buffer at room temperature for 5 minutes
Washed with high stringency wash buffer 2 times for 20 minutes at room temperature

NOTE: Because bacterial colonies were lifted on membranes from plates that had been prepared for IPTG/X-gal blue/white screening there were many blue spots on the membranes even before the colorimetric procedure was started.  To account for this the membranes were photographed both before and after the colorimetric procedure
(It is a good idea NOT to use IPTG/X-gal blue/white screening on the plates that will be used for colorimetric screening later!)

Colorimetric detection of DIG labeled nucleic acids was performed following the instructions provided with the DIG Nucleic Acid Detection Kit purchased from Roche Molecular Biochemicals

Selection of Colonies for Plasmid Extraction
Colonies that had a very dark purple color were considered to be positive (containing iguana DNA within the plasmid with a microsatellite repeat sequence)
Positive colonies were identified and then grown up in overnight cultures for plasmid DNA extraction

Overnight cultures contained 4 ml sterile autoclaved LB broth with a sterile toothpick that had been touched to a selected positive bacteria colony added (using sterile procedure)
Incubation was at 37^oC overnight on an orbital shaker at 200 RPM

Plasmid DNA extraction was done using QIAGEN's QIAprep Spin Miniprep Kit
Approximate DNA concentration was determined by agarose gel electrophoresis with a known quantitative DNA ladder using the "spot density" function included with the IS-1000 Digital Imaging System used for documentation

In order to select plasmids with inserted DNA to sequence that would provide the most valuable information a "secondary screening" procedure was performed.  The principles behind this procedure may be examined on a separate page.  An overview of the secondary screening procedure is available as a pop-up window:
 

Procedural Details:

Ran 2 separate PCR reactions for each extracted DNA sample

1.   PCR using the same primers used when sequencing reactions are performed - "sequencing primers"  (See this site for an overview of pUC19 and where DNA was inserted [at the BamH1 site], that shows where the "sequencing primers" bind.)
2.   Another PCR using the "sequencing primers" along with all 6 of the synthesized (unlabeled) oligos previously used for hybridization and detection of microsatellite repeat sequences

PCR was performed using the following recipe prepared on ice (for 9 10 µl reactions): [Mayo 18]

54 µl [46 µl - if additional oligos used] PCR grade water
7.5 µl MgCl₂ (25 mM stock) - 2 mM final concnetration¹
9 µl 10x Buffer¹
9 µl dNTP's²
0.5 µl upper "sequencing primer" (21M13)^3,5
0.5 µl lower "sequencing primer" (Reverse 2)^3,5
[if being used - 1.3 µl of each of the 6 synthesized oligos^4,5 containing microsatellite repeats - 7.8 µl]
1 µl Taq¹

¹ Provided with Promega Taq DNA Polymerase
² Purchased from Epicentre as a premixed stock solution containing 2.5 mM of each dNTP
³ Diluted in TE to a concentration of 250 pmol/µl
⁴ Diluted in TE to a concentration of 100 pmol/µl
⁵ All oligonucleotide primers synthesized by Genosys

9 µl of the above "master mix" and 1 µl plasmid DNA were put into each sample tube
Each sample was then covered with 20 µl mineral oil (to prevent evaporation during PCR)
PCR was run using the following program: [Junio 7]

45 cycles of . . .

94^oC for 45 seconds
60^oC for 1 minute
72^oC for 1 minute

72^oC for 9 more minutes
Cooled down and held at 4^oC

PCR Products were run as pairs on an 8% polyacrylamide (16 cm long) gel for 1.5 hours at 300 V [Mayo 19, 26]

Pairs in which the PCR sample containing the oligonucleotide repeat sequences as primers had a shorter product
clearly visible were considered positive - indicating that a repeat sequence was located within the inserted DNA of the plasmid.  (See the additional page containing an explanation of the reasoning behind the procedure.)

Selection of Plasmids to Send Out for Sequencing:

PCR data for each extracted plasmid were entered into a spreadsheet and sorted by total length of DNA insert (the size of the product produced when only "sequencing primers" were used).  Plasmids that had products between 300-1000 were considered preferable (since it is fairly certain that sequencing in both direction will cover the entire inserted DNA sequence), however up to 1500 was considered OK.  Those DNA inserts that looked like the repeat sequence was in the middle (i.e. they had a shorter PCR product that was about half the size of the entire insert) or at least 100-200 bp from either end of the insert were believed to have the highest probability of yielding good sequence data that would allow ample room on either side of the microsatellite repeat to design functional primers.

Plasmids chosen for sequencing were dried down in a known quanitity using a speedvac (centrifuge with a vacuum applied) and sent to the sequencing facility at California State University - Northridge

Design and Testing of Primers:

PCR primers were designed from the sequences received using Oligo 4.0 from Molecular Biology Insights.  The things to look for in selection of primers are:

• Small difference in template-primer T_m, less that 35^oC

(T_m is the temperature at which 50% of a nucleic acid is in duplex with a complementary strand.)
• Relatively low 3'-terminal stability, so that the 3'-pentamer delta-G is more positive than -9 kcal/mol

(delta-G is a measurement of free energy.  DNA is more stable [held together more tightly with more stable bonds] when delta-G is more negative.)
 
• A higher proportion of GC-content on the 5'-end of the primer is desirable
• Small difference between the T_m of the primers

(A large difference in T_m between primers can lead to the primer with the higher T_m binding unspecifically at the temperature necessary for the other primer to bind.)
• Dimer formation (binding) between primers and themselves or to each other is bad, especially when the delta-G value is quite negative
• Hairpin formation (a primer folding around and binding to itself) is bad, especially when the delta-G value is quite negative
• Primers with approximately 50% GC-content are preferred
• T at the 3'-end may lead to more frequent mispriming than A, G, or C
• Since low annealing temperatures are more likely to produce non-specific products primers with higher melting temperatures allowing high annealing temperatures are beneficial
• It is best to design primers as close to the microsatellite repeat as possible.  This reduces the size of the PCR product(s) produced and makes separation and analysis of the product(s) on gels faster and more accurate.

Each set of developed primers was optimized for PCR product specificity (so there weren't a lot of extra "junk" product bands) by altering PCR annealing temperature and MgCl₂ concentration.

Ampicillin Solution:

100 mg/ml ampicillin
In distilled water
Sterile filtered through  0.22 micron filter

Calcium Chloride Solution:

0.05 M CaCl₂
In distilled water
Sterile filtered through  0.22 micron filter

Denaturing Solution:

0.5 M NaOH
1.5 M NaCl
In distilled water

IPTG Solution:

100 mg/ml isopropyl-[beta]-D-thiogalactopyranoside
In distilled water

Lithium Chloride Solution:

4 M LiCl
In distilled water
Sterile filtered through  0.22 micron filter

Neutralizing Solution:

1.5 M NaCl
0.5 M Tris-HCl
In distilled water
pH adjusted to 7.4

20x SSC:

3 M NaCl
0.3 M Sodium citrate
In distilled water
pH adjusted to 7.0

Tris-EDTA (TE) Buffer:

0.01 M Tris-HCl
0.001 M EDTA
pH adjusted to 8.0
Sterile filtered through 0.22 micron filter

Tris-HCl Buffer:

0.05 M Tris-HCl
pH adjusted to 7.2
Sterile filtered through 0.22 micron filter

Wash Buffers - Low / High Stringency:

Low 2x SSC / High 0.5x SSC
0.1% SDS Low / High
In distilled water

X-gal Solution:

20 mg/ml 5-bromo-4-chloro-3-indolyl-[beta]-D-galactopyranoside
In dimethylformamide

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