Handling of NIA 15K cDNA clone set (June 3, 2000)

1. There are 159 plates (96-well). Plate IDs are: H3001 - H3159. Each cDNA clones are named H3001A01 - H3159H12 according to their well positions. 3’-end sequences of these cDNA clones are named H3001A01-3, etc.
   
   
2. All cDNAs are cloned in NotI/SalI site of Ampicillin-resistant pSPORT1 vector (Life Technologies). Average insert size of the clones is 1.5 kb (0.5 - 3 kb).
   
   
3. E. coli host strain is DH10B.
   
   
4. Individual cDNA clones are grown in 2 XYT media including glycerol.
   
   

Protocol for 2XYT Medium (with Glycerol)

• Mix with water until all reagents is completely dissolved. Adjust pH to 7.0. -dilute to 1.0 liter with water.
  
  
• Autoclave (note: all reagents must be autoclaved)
  
  

Before using add: per 1.0 ml of 2XYT media

For various reasons (e.g., clones did not grow), there are 182 wells that do not contain bacteria (medium only).

H3001E06	H3053C10	H3099G04	H3124C01	H3145C05
H3001H02	H3053E09	H3101F01	H3124C02	H3145C06
H3011D08	H3055B01	H3101G01	H3124C03	H3145C07
H3011E08	H3055B03	H3101G08	H3124C04	H3145C08
H3011E12	H3058F12	H3101H10	H3124C05	H3145C09
H3011F04	H3059C09	H3101H12	H3124C06	H3145C10
H3019D06	H3059H08	H3103A03	H3124C07	H3145C11
H3023G11	H3062G01	H3104G08	H3124D10	H3145C12
H3023H01	H3064A04	H3106B03	H3124E10	H3145D01
H3023H02	H3064H10	H3106F11	H3125H09	H3145D02
H3023H03	H3067D04	H3107E07	H3126A02	H3145D03
H3023H04	H3069E10	H3107H02	H3126A07	H3145D04
H3023H05	H3070F12	H3107H12	H3126A08	H3145D05
H3023H06	H3071A01	H3112C07	H3126B03	H3145D06
H3023H07	H3071A10	H3112C08	H3126B05	H3145D07
H3023H08	H3071D10	H3114A03	H3126G01	H3145D08
H3024A08	H3072C11	H3114G03	H3126G04	H3145D09
H3025B07	H3074A12	H3114G04	H3126G11	H3150A11
H3026A07	H3074E05	H3114G05	H3130H07	H3151H03
H3032E02	H3074G09	H3114G06	H3130H08	H3152E09
H3032E05	H3075F04	H3114G07	H3132D10	H3156E11
H3032F01	H3076C11	H3114G08	H3132H10	H3157A08
H3035C04	H3078B06	H3114G09	H3139D04
H3036C04	H3081A03	H3114G10	H3139D08
H3041B12	H3083H01	H3114G11	H3139E05
H3041F01	H3084E02	H3115D11	H3139E09
H3042C12	H3086A05	H3115D12	H3139E11
H3042F02	H3086H07	H3115E01	H3139F10
H3043A08	H3087B05	H3115H05	H3140G07
H3043G06	H3093A03	H3116F12	H3140G10
H3043H01	H3093B08	H3117C07	H3140G11
H3044B03	H3093C07	H3118C04	H3140H06
H3046A02	H3096F05	H3119B07	H3145B09
H3048E01	H3097C05	H3119C07	H3145B10
H3049A02	H3097E09	H3120A09	H3145B11
H3049A03	H3098D11	H3120A10	H3145B12
H3050E06	H3098E08	H3120G08	H3145C01
H3050E07	H3098F01	H3120H08	H3145C02
H3052E03	H3098F02	H3120H09	H3145C03
H3053B01	H3098F03	H3122G04	H3145C04

Preparation of DNAs for microarray

Plasmid DNAs were prepared in 96-well format (R.E.A.L. Prep 96 plasmid kit; Qiagen Inc., Valencia CA). To amplify cDNA inserts,

PCR primers ( 5’-CCAGTCACGACGTTGTAAAACGAC-3’; 5’-GTGTGGAATTGTGAGCGGATAACAA-3’)

were designed from sequences flanking the multiple cloning site of pSPORT1 plasmid vector (Life Technologies, Rockville, MD), which was used for cDNA cloning. For each cDNA clone, about 10 ng of plasmid DNA was used in 100 µl PCR reaction containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.5 µM each primer, 0.8 mM dNTPs and 3.25 units of Taq polymerase (PE Biosystems, Foster City, CA). Amplification was done in a Peltier Thermal Cycler (MJ Research Inc., Waltham, MA) under the following condition: initial denaturation at 95°C for 1 min; 38 cycles of denaturation at 94°C for 30 seconds, annealing at 65°C for 45 seconds, and extension at 72°C for 3.5 min; and a final extension at 72 °C for 3 min. PCR products were precipitated with 50% isopropanol, washed in 70% ethanol and resuspended in 6.5 µl TE buffer (pH 8.0). To examine the quality and quantity of PCR products, they were run on 1% agarose gels (Embi Tec, San Diego, CA). Greater than 90% of the clones gave single PCR products.

For further information, please check the following Web Site

Contact:

Minoru S. H. Ko, M.D./Ph.D.
Developmental Genomics & Aging Section
Laboratory of Genetics, National Institute on Aging
National Institutes of Health
333 Cassell Drive, Suite 4000
Baltimore, MD 21224-6825, USA
Phone : 410-558-8359
FAX: 410-558-8331
E-mail: KoM@grc.nia.nih.gov