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Project Documentation & Protocols: Maize Gene Discovery Project: Microarrays: Libraries

Contents: Index | Progress | Controls | Libraries | Arrays | Data | Protocols | Ordering | Links | FAQs

Library Elements Search
This page allows you to search for information about all ESTs in each EST library that has been printed on any microarray slide from the Maize Gene Discovery Project. The information returned will include the current TUC (tentative unique contig) to which that EST belongs and a putative protein identification through Blastx. You can search for any word by typing it into the "text search" box below. You can also specify the size of EST printed by entering the length criteria (be sure to use the pull down menu to specify which array type). If you simply want to browse through all of the records, click the "Search" button.

Format:
General text search in all fields:
Specific EST length criteria (bp)

Microarray Format Description

Microarray format 605.03
Microarray format 605.04
Microarray format 606.01
Microarray format 614.01
Microarray format 486

Microarray Element Table 1.0 Print 605.03.01: Project 605 Developing Endosperm /Microarray Format 03/Print 01

Format 03 Differs from 04

  • Printed as 2 Duplicate Arrays each with No Replicate Spots.
  • Printed with 4 pin configuration
  • Sample Plate 91 was missing from printing

PCR Amplification, Product Analysis, and Clean-up

All PCR reactions were carried out in 96-well plates. Samples were amplified in two separate PCR reactions (a 50 µl and a 75 µl reaction). Final reaction concentrations were as follows: (DNTP 0.2 mM, primers 0.2 um, Sigma Taq Polymerase {Sigma D1806} 2.25 units per 75 µl reaction {used with 10 X Reaction Buffer containing 1.5 mM MgCl2}). Primers used for this library were 5'-C6 amino modified (sequence given 5'-3'; forward: CTG CAG TAA TAC GAC TCA CTA TAG; reverse: CTA TTC GAT GAT GAA GAT ACC). One to 2 µl of template DNA (purified plasmid DNA received from Stanford) was added to the 96-well PCR plate containing reaction mix using a sterile 96-well replicating tool. Reaction conditions were as follows: 94°C for 2 minutes; then 40 cycles of 94°C for 30 sec, 55C for 30 sec, 72°C for 1 min; then 72°C for 10 min and hold at 4°C. All products were analyzed by gel electrophoresis (1.2% agarose gels, 2 µl of PCR product). Plates were cleaned-up using Millipore Multiscreen PCR Plates (Millipore MANU 030 50) according to manufacturer's instructions. The cleaned-up products were stored in V-Bottom 96-well plates (Greiner 651 180) for printing. Plates were dried using a Savant Speedvac Concentrator (60 °C,2 hrs) and stored dry at -20 C.

Preparation for Printing:

Plates were resuspended in 15 µl of 2X SSC at room temperature at least 1 hr prior to printing. If not used immediately after resuspension period, plates were stored at 4 °C and placed at room temperature at least 15 min prior to printing. After printing plates were dried (60 °C,20 min) in Speedvac concentrator and stored at -20 °C.

Printer Configuration:

Microarray slides were printed using an OmniGridder (GeneMachines, San Carlos, California) and 4 Majer Precision pins (11077-1 Rev A MicroQuill 2000 Microarray Printing Tip, Majer Precision Engineering, Tempe, Arizona). We printed 100 slides each printing. Instrument settings were as follows: Dip time 1000 milliseconds, Print time 0, After every Sample (Wash 2000ms, Dry 2000ms) 8 times, After every Plate (Sonicate 30,000ms, Wash 10,000ms, Dry 2000ms) 2 times-wash solution was nanopure water. Instrument settings were optimized prior to printing using a Cy3-labeled oligo to check print quality and verify no carryover between samples.

Print Design:

Slide Format:

The 605 Library (plus controls) was printed as 2 duplicate arrays each with no replicate spots. Each slide is composed of eight subarray--each subarray is printed by a single pin. When a slide is oriented with the label down (text of label is in correct orientation) the subarrays are positioned in 4 Rows and 2 columns. Printing the slide with 4 pins in a 2 x 2 configuration, produces duplicate arrays each composed of 4 subarrays in a 2 x2 configuration. Thus subarrays in Row 1 Column 1 and Row 3 Column 1 are printed with the same pin and are identical (likewise subarrays in Row 2 Column 1 and Row 4 Column 1 are identical, subarrays in Row 1 Column 2 and Row 3 Column 2 are identical, subarrays Row 2 Column 2 and Row 4 Column 2 are identical).

Subarray Format

Each of the 8 subarrays is composed of a 46 x 46 matrix of spots-together there are 2116*8=16,926 spot positions on a slide. Spots were printed at a spacing of 190 um with a 380 um gap between subarrays. We printed a total of 83 different sample plates containing 96 samples each and 93 different controls. Controls and 2 sample plates were printed twice per replicate array. Samples and controls combined to produce 8346 spots of DNA per array x 2 arrays per slides = 16,692 spots of DNA on a slide-remaining spot positions were blank. Each DNA element on the slide can be located by knowing the Row and Column of the Subarray and the Row and Column within the Subarray where the element is printed. The printed area of the slide is 18 X 36 mm. Experimenters will need to use 22 X 40 coverslips for hybridization (e.g. Sigma Z36,591-2).

Slide Source and Storage After Printing:

Microarrays were printed on Sigma Screen Silane Slides (Sigma S7934-50EA). Slides were stored without immobilizing elements in slide containers at room temperature in the dark.

Microarray Element Table 2.0 Print 605.04.01 & 02: Project 605 Developing Endosperm /Microarray Format 04/Prints 01 and 02

Format 03 Differs from 04

  • Printed as a Single Array with 2 Replicate Spots (side-by-side) of each element.
  • Printed with 8 pin configuration
  • Sample plate 91 was printed
  • Dip time was decreased to 500 ms and 0.5 X SSC was used in wash cycle on print 02.

PCR Amplification, Product Analysis, and Clean-up:

Used same samples as for the 605.03 printing (see text 605.03 for Methods).

Preparation for Printing:

After each printing the plates were dried using a Speedvac concentrator (60 °C,20 min). Prior to printing, the plates were resuspended in 15 µl of sterile distilled water at room temperature at least 1 hr prior to printing. If not used immediately after resuspension period, plates were stored at 4 °C and placed at room temperature at least 15 min prior to printing. After printing plates were again dried (60 °C,20 min) in Speedvac concentrator and stored at -20 °C.

Printer Configuration:

Microarray slides were printed using an OmniGridder (GeneMachines, San Carlos, California) and 8 Majer Precision pins (11077-1 Rev A MicroQuill 2000 Microarray Printing Tip, Majer Precision Engineering, Tempe, Arizona). We printed 100 slides each printing. Instrument settings were as follows: Dip time 500 milliseconds, Print time 0, After every Sample (Wash 2000ms, Dry 2000ms) 8 times, After every Plate (Sonicate 30,000ms, Wash 10,000ms, Dry 2000ms) 2 times-wash solution was 0.5 X SSC. Instrument settings were optimized prior to printing using a Cy3-labeled oligo to check print quality and verify no carryover between samples.

Print Design:

The 605 Library (plus controls) was printed in a single array with two replicate spots of each element-replicate spots were printed side-by-side. Each slide is composed of eight subarrays each printed by a separate pin. When a slide is oriented with the label down (text of label is in correct orientation) the subarrays are positioned in 4 Rows and 2 columns. Because the slide is printed with 8 pins in a 4 x 2 configuration, each subarray is printed with a different pin.

Subarray Format

Each of the 8 subarrays is composed of a 46 x 46 matrix of spots-together there are 2116*8=16,926 spot positions on a slide. Spots were printed at a spacing of 190 um with a 380 um gap between subarrays. We printed a total of 84 different sample plates containing 96 samples each and 93 different controls. Sample plates were printed once and control plates were printed twice. Samples and controls combined to produce 8250*2 = 16,500 spots of DNA on a slide-remaining spot positions were blank. Each DNA element on the slide can be located by knowing the Row and Column of the Subarray and the Row and Column within the Subarray where the element is printed. The printed area of the slide is 18 X 36 mm. Experimenters will need to use 22 X 40 coverslips for hybridization (e.g. Sigma Z36,591-2).

Slide Source and Storage After Printing:

Microarrays were printed on Sigma Screen Silane Slides (Sigma S7934-50EA). Slides were stored without immobilizing elements in slide containers at Room Temperature in the dark.

Microarray Element Table 3.0 Print 606.01.01: Project 606 Ear Tissue /Microarray Format 01/Print 01-03

Plate Consolidation

Prior to PCR amplification, the 606 EST library was consolidated by the removal of sequences with a DNP code (Do Not Print code-generally given for small or redundant sequences). We used a Beckman Coulter Biomek 2000 liquid handling robot to 'cherry-pick' aliquots of desired sequences from source plates (purified plasmid DNA received from Stanford) to new plates that would serve as our template source for PCR amplification. The Biomek removed a 5 µl aliquot (1/10 volume) of each EST using sterile tips. We then diluted each aliquot with 15 µl of sterile water and stored the new plates at -20 °C until required for PCR amplification. Original Stanford plates were also returned to storage at -20 C.

PCR Amplification, Product Analysis, and Clean-up

All PCR reactions were carried out in 96-well plates. Samples were amplified in two separate 75 µl PCR reactions. Final reaction concentrations were as follows: (DNTP 0.2 mM, primers 0.2 um, Sigma Taq Polymerase {Sigma D1806} 2.25 units per 75 µl reaction {used with 10 X Reaction Buffer containing 1.5 mM MgCl2}). Primers used for this library were 5'-C6 amino modified (sequence given 5'-3'; forward: GTA ATA CGA CTC ACT ATA GGG C; reverse: AAT TAA CCC TCA CTA AAG GG). One to 2 µl of template DNA was added to the 96-well PCR plate containing reaction mix using a sterile 96-well replicating tool. Reaction conditions were as follows: 94°C for 2 minutes; then 40 cycles of 94°C for 30 sec, 55C for 30 sec, 72°C for 1 min; then 72°C for 10 min and hold at 4°C. All products were analyzed by gel electrophoresis (1.2% agarose gels, 2 µl of PCR product). Plates were cleaned-up using Millipore Multiscreen PCR Plates (Millipore MANU 030 50) according to manufacturer's instructions. The cleaned-up products were stored in V-Bottom 96-well plates (Greiner 651 180) for printing. Plates were dried using a Savant Speedvac Concentrator (60 °C,2 hrs) and stored dry at -20 C.

Preparation for First Printing:

Plates were resuspended in 15 µl of 2X SSC at room temperature at least 1 hr prior to printing. If not used immediately after resuspension period, plates were stored at 4 °C and placed at room temperature at least 15 min prior to printing. After printing plates were dried (60 °C,20 min) in Speedvac concentrator and stored at -20 °C.

Preparation for Second and Third Printing:

After each printing, the plates were dried using a Speedvac concentrator (60 °C,20 min). Prior to reprinting, the plates were resuspended in 15 µl of sterile distilled water at room temperature at least 1 hr prior to printing. If not used immediately after resuspension period, plates were stored at 4 °C and placed at room temperature at least 15 min prior to printing. After printing plates were again dried (60 °C,20 min) in Speedvac concentrator and stored at -20 °C.

Printer Configuration:

Microarray slides were printed using an OmniGridder (GeneMachines, San Carlos, California) and 8 Majer Precision pins (11077-1 Rev A MicroQuill 2000 Microarray Printing Tip, Majer Precision Engineering, Tempe, Arizona). We printed 100 slides each printing. Instrument settings were as follows: Dip time 500 milliseconds, Print time 0, After every Sample (Wash 2000ms, Dry 2000ms) 8 times, After every Plate (Sonicate 30,000ms, Wash 10,000ms, Dry 2000ms) 2 times-wash solution was 0.5 X SSC. Instrument settings were optimized prior to printing using a Cy3-labeled oligo to check print quality and verify no carryover between samples.

Print Design:

Slide Format:

The 606 Library (plus controls) is printed as a single array with 3 replicate spots of each element. Each slide is composed of eight subarrays each printed by a separate pin. When a slide is oriented with the label down (text of label is in correct orientation) the subarrays are positioned in 4 Rows and 2 columns. Because the slide is printed with 8 pins in a 4 x 2 configuration, each subarray is printed with a different pin.

Subarray Format

Each of the 8 subarrays is composed of a 45 x 45 matrix of spots-together there are 2025*8=16,200 spot positions. Spots were printed at a spacing of 195 um with a 250 um gap between subarrays. We printed 52 different sample plates containing a total of 4980 samples and 2 control plates containing a total of 113 different controls. Sample plates were printed once and control plates were printed twice--each element was printed in triplicate. Samples and controls combined to produce 5206*3=15,618 spots of DNA on a slide--remaining spot positions were blank. Each DNA element on the slide can be located by knowing the Row and Column of the Subarray and the Row and Column within the Subarray where the element is printed. The printed area of the slide is 18 X 36 mm. Experimenters will need to use 22 X 40 coverslips for hybridization (e.g. Sigma Z36,591-2).

Slide Source and Storage After Printing:

Microarrays were printed on Sigma Screen Silane Slides (Sigma S7934-50EA). Slides were stored without immobilizing elements in slide containers at room temperature in the dark.

Microarray Element Table 4.0 Print 614.01.01: Project 614 Root Tissue /Microarray Format 01/Print 01-03.

Plate Consolidation

Prior to PCR amplification, the 614 EST library was consolidated by the removal of sequences with a DNP code (Do Not Print code-generally given for small or redundant sequences). We used a Beckman Coulter Biomek 2000 liquid handling robot to 'cherry-pick' aliquots of desired sequences from source plates (purified plasmid DNA received from Stanford) to new plates that would serve as our template source for PCR amplification. The Biomek removed a 5µl aliquot (1/10 volume) of each EST using sterile tips. We then diluted each aliquot with 15 µl of sterile water and stored the new plates at -20 °C until required for PCR amplification. Original Stanford plates were also returned to storage at -20 °C.

PCR Amplification, Product Analysis, and Clean-up

All PCR reactions were carried out in 96-well plates. Samples were amplified in two separate 75 µl PCR reactions. Final reaction concentrations were as follows: (DNTP 0.2 mM, primers 0.2 um, Sigma Taq Polymerase {Sigma D1806} 2.25 units per 75 µl reaction {used with 10 X Reaction Buffer containing 1.5 mM MgCl2}). Primers used for this library were 5'-C6 amino modified (sequence given 5'-3'; forward: GTA ATA CGA CTC ACT ATA GGG C; reverse: AAT TAA CCC TCA CTA AAG GG). One to 2 µl of template DNA was added to the 96-well PCR plate containing reaction mix using a sterile 96-well replicating tool. Reaction conditions were as follows: 94°C for 2 minutes; then 40 cycles of 94°C for 30 sec, 54°C for 30 sec, 72°C for 1:30 min; then 72°C for 10 min and hold at 4°C. All products were analyzed by gel electrophoresis (1.2% TAE agarose gels, 2 µl of PCR product). Plates were cleaned-up using Millipore Multiscreen PCR Plates (Millipore MANU 030 50) according to manufacturer's instructions. The cleaned-up products were stored in V-Bottom 96-well plates (Greiner 651 180) for printing. Plates were dried using a Savant Speedvac Concentrator (60 °C,2 hrs) and stored dry at -20 °C.

Preparation for First Printing:

Plates were resuspended in 15 µl of 3X SSC at room temperature at least 1 hr prior to printing. If not used immediately after resuspension period, plates were stored at 4 °C and placed at room temperature at least 15 min prior to printing. After printing plates were dried (60 °C,20 min) in Speedvac concentrator and stored at -20 °C.

Preparation for Second and Third Printing:

After each printing, the plates were dried using a Speedvac concentrator (60 °C,20 min). Prior to reprinting, the plates were resuspended in 15 µl of sterile distilled water at room temperature at least 1 hr prior to printing. If not used immediately after resuspension period, plates were stored at 4 °C and placed at room temperature at least 15 min prior to printing. After printing plates were again dried (60 °C,20 min) in Speedvac concentrator and stored at -20 °C.

Printer Configuration:

Microarray slides were printed using an OmniGridder (GeneMachines, San Carlos, California) and 8 Majer Precision pins (11077-1 Rev A MicroQuill 2000 Microarray Printing Tip, Majer Precision Engineering, Tempe, Arizona). We printed 100 slides each printing. Instrument settings were as follows: Dip time 500 milliseconds, Print time 0, After every Sample (Wash 2000ms, Dry 2000ms) 8 times, After every Plate (Sonicate 30,000ms, Wash 10,000ms, Dry 2000ms) 2 times-wash solution was 0.5 X SSC. Instrument settings were optimized prior to printing using a Cy3-labeled oligo to check print quality and verify no carryover between samples.

After each sample pick up, the pins visited a glass blotting pad. The blotting pad is designed to allow the pins to print several times on a blotter before striking the first slide. Typically spot quality is poor for the first 1-20 pin strikes-the goal is to minimize the number of first slides with poor spots quality by printing the first spots on the blotting pad. For print 01, the pins preprinted 1 time on the blotter. For print 02 and 03, the pins preprinted 10 times on the blotter.

Print Design:

Slide Format:

The 614 Library (plus controls) is printed as a single array with 3 replicate spots of each element. Each slide is composed of eight subarrays each printed by a separate pin. When a slide is oriented with the label down (text of label is in correct orientation) the subarrays are positioned in 4 Rows and 2 columns. Because the slide is printed with 8 pins in a 4 x 2 configuration, each subarray is printed with a different pin.

Subarray Format

Each of the 8 subarrays is composed of a 45 x 45 matrix of spots-together there are 2025*8=16,200 spot positions. Spots were printed at a spacing of 195 um with a 1000 um gap between subarrays. We printed 53 different sample plates containing a total of 5065 samples and 2 control plates containing a total of 116 different controls. Sample plates were printed once and control plates were printed twice--each element was printed in triplicate. Samples and controls combined to produce 5297*3=15,891 spots of DNA on a slide--remaining spot positions were blank. Each DNA element on the slide can be located by knowing the Row and Column of the Subarray and the Row and Column within the Subarray where the element is printed. The printed area of the slide is 18 X 36 mm. Experimenters will need to use 22 X 40 coverslips for hybridization (e.g. Sigma Z36,591-2).

Slide Source and Storage After Printing:

Microarrays were printed on Sigma Screen Silane Slides (Sigma S7934-50EA). Slides were stored without immobilizing elements in slide containers at room temperature in the dark.

Microarray Element Table 5.0 Print 486.01.01: Project 486 Immature Leaf/Microarray Format 01/Print 01

Plate Consolidation

Prior to PCR amplification, the 486 EST library was consolidated by the removal of sequences with a DNP code (Do Not Print code-generally given for small or redundant sequences). We used a Beckman Coulter Biomek 2000 liquid handling robot to 'cherry-pick' aliquots of desired sequences from source plates (purified plasmid DNA received from Stanford) to new plates that would serve as our template source for PCR amplification. The Biomek removed a 5l aliquot (1/10 volume) of each EST using sterile tips. We then diluted each aliquot with 15 l of sterile water and stored the new plates at -20 C until required for PCR amplification. Original Stanford plates were also returned to storage at -20 C.

PCR Amplification, Product Analysis, and Clean-up

All PCR reactions were carried out in 96-well plates. Samples were amplified in two separate 75 l PCR reactions. Final reaction concentrations were as follows: (DNTP 0.2 mM, primers 0.2 um, Sigma Taq Polymerase {Sigma D1806} 2.25 units per 75 l reaction {used with 10 X Reaction Buffer containing 1.5 mM MgCl2}). Primers used for this library were 5'-C6 amino modified (sequence given 5'-3'; forward: GTA ATA CGA CTC ACT ATA GGG C; reverse: AAT TAA CCC TCA CTA AAG GG). One to 2 l of template DNA was added to the 96-well PCR plate containing reaction mix using a sterile 96-well replicating tool. Reaction conditions were as follows: 94C for 2 minutes; then 40 cycles of 94C for 30 sec, 54C for 30 sec, 72C for 1:30 min; then 72C for 10 min and hold at 4C. All products were analyzed by gel electrophoresis (1.2% TAE agarose gels, 2 l of PCR product). Plates were cleaned-up using Millipore Multiscreen PCR Plates (Millipore MANU 030 50) according to manufacturer's instructions. The cleaned-up products were stored in V-Bottom 96-well plates (Greiner 651 180) for printing. Plates were dried using a Savant Speedvac Concentrator (60 C,2 hrs) and stored dry at -20 C.

Preparation for First Printing:

Plates were resuspended in 15 l of 2X SSC at room temperature at least 1 hr prior to printing. If not used immediately after resuspension period, plates were stored at 4 C and placed at room temperature at least 15 min prior to printing. After printing plates were dried (60 C,20 min) in Speedvac concentrator and stored at -20 C.

Preparation for Second and Third Printing:

After each printing, the plates were dried using a Speedvac concentrator (60 C,20 min). Prior to reprinting, the plates were resuspended in 15 l of sterile distilled water at room temperature at least 1 hr prior to printing. If not used immediately after resuspension period, plates were stored at 4 C and placed at room temperature at least 15 min prior to printing. After printing plates were again dried (60 C,20 min) in Speedvac concentrator and stored at -20 C.

Printer Configuration:

Microarray slides were printed using an OmniGridder (GeneMachines, San Carlos, California) and 8 Majer Precision pins (11077-1 Rev A MicroQuill 2000 Microarray Printing Tip, Majer Precision Engineering, Tempe, Arizona). We printed 100 slides each printing. Instrument settings were as follows: Dip time 500 milliseconds, Print time 0, After every Sample (Wash 2000ms, Dry 2000ms) 8 times, After every Plate (Sonicate 30,000ms, Wash 10,000ms, Dry 2000ms) 2 times-wash solution was dH2O. Instrument settings were optimized prior to printing using a Cy3-labeled oligo to check print quality and verify no carryover between samples.

After each sample pick up, the pins visited a glass blotting pad. The blotting pad is designed to allow the pins to print several times on a blotter before striking the first slide. Typically spot quality is poor for the first 15 pin strikes-the goal is to minimize the number of first slides with poor spots quality by printing the first spots on the blotting pad. For print 01, the pins preprinted 1 time on the blotter. For print 02 and 03, the pins preprinted 10 times on the blotter.

Print Design:

Slide Format:

The 486 Library (plus controls) is printed as a single array with 3 replicate spots of each element. Each slide is composed of eight subarrays each printed by a separate pin. When a slide is oriented with the label down (text of label is in correct orientation) the subarrays are positioned in 4 Rows and 2 columns. Because the slide is printed with 8 pins in a 4 x 2 configuration, each subarray is printed with a different pin.

Subarray Format

Each of the 8 subarrays is composed of a 41(rows) x 45 (columns) matrix of spots-together there are 1845*8=14760 spot positions. Spots were printed at a spacing of 200 um with a 1000 um gap between subarrays. We printed 47 different sample plates containing a total of 4454 samples and 2 control plates containing a total of 116 different controls. Sample plates were printed once and control plates were printed twice--each element was printed in triplicate. Samples and controls combined to produce 4570*3=13,710 spots of DNA on a slide--remaining spot positions were blank. Each DNA element on the slide can be located by knowing the Row and Column of the Subarray and the Row and Column within the Subarray where the element is printed. The printed area of the slide is 18 X 36 mm. Experimenters will need to use 22 X 40 coverslips for hybridization (e.g. Sigma Z36,591-2).

Slide Source and Storage After Printing:

Microarrays were printed on Clonetech Silane Slides (DNA-Ready Type-I slides, Cat#7880-2). Slides were stored without immobilization at room temperature in the dark.


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