Additional Notes on cDNA Library Preparations
NB: These are additional notes, mostly from Carolina Livi, that supplement the Gibco-BRL Superscript cDNA library kit manual. The plasmid vector is called pSPORT. This description specifically pertains to the embryonic libraries prepared in the Davidson laboratory (7hr, 15hr, 20hr, 30hr, 40hr). The other cDNA libraries are all in pSport [Sequence] but the details of preparation may differ in the Livingston and Ettensohn laboratories.
Total RNA. we use RNAzol TM B from Leedo Medical Laboratories Catalog CS-104 for 100 ml or Catalog CS-105 for 200ml. We follow the protocol attached with it.
mRNA Isolation. To isolate mRNA you will need Dynabeads Oligo (dT)25 from Dynal(phone 800 638 9416) Product No. 610.02 for 2 ml or 610.05 for 5 ml.
Library. he kit we use to make the library is SuperScript TM Plasmid System for cDNA Synthesis and Plasmid Cloning from GibcoBRL Catalog 18248-013 (you can make 3 libraries with the kits, but in my experience you run out of tRNA, which can be purchases separately or you can use some you have hanging around). Instead of using the column from the Gibco kit, we used one provided by cDNA Synthesis Kit from Stratagene Catalog 200401.
RNA Isolation. Here is the procedure we use to isolate mRNA with the Dynabeads. Basically we have combined many of the protocols suggested on their information sheet.
mRNA isolation with Dynabead (oligo dT) from total RNA
1) Dispense 600ul of beads, wash with 2X BINDING BUFFER.
2) add 300 ul 2X BINDING BUFFER + 300ul (225ug) total RNA (0.75ug/ul).
3) wash 3X with 600ul 1X WASHING BUFFER.
4) elute with 60ul of RNase free water.
5) wash the beads with 600ul of 1X WASHING BUFFER 2 times.
6) wash beads with 300 ul of 2X BINDING BUFFER
7) repeat steps 2-6,4 times.
8) precipitate mRNA add to the 300 ul mRNA in ddH2O obtained above:
30ul 3M NaOAc
660ul 100% EtOH
keep at -20oC O/N or until needed
Approximate yield: 30ug mRNA / 1000ug total RNA Before using mRNA from library, run ~ 1ug in agarose gel (boil the mRNA for no longer than 3 min). The recipe for the BINDING and WASHING BUFFERS is in the Dynabeads information sheet.
For the cDNA library one should start with 5 ug of mRNA. We followed the standard protocol from the kit except we used our own primers (see below) not those provided with the kit. We add the SuperScript II enzyme at room temperature instead of at 37°C. We get between 5X 10E5 - 5 X 10E6 clones when we start with 5 ug of mRNA.
It's the ElectroMAX DH10B TM cells from GibcoBRL Catalog 18290-015 (size 0.5ml). This item is often backordered and should be ordered in advance. The bacteria should not be stored for a long time (more than 3 months) for the purpose of the libraries(although leftovers can be used for other applications). The 500 ul will probably be enough for 1 library, but if you have other uses for them I would have 1 ml available. We follow the transformation procedure provided in the information sheet.
Storage of libraries
We usually freeze (-70) the transformed bacteria (after plating 10ul of a 1 ml aliquot to check the transformation efficiency and colony density. We tend to get at least 90% of the colony density after thawing, so we don't find this a minus.
Below is the description of the primers we used to make our libraries.
A A A G G A A G G A A A A A A G C G G C C G C T A C A N(8) T
XO Block Not I site marker random
The sequence marked XO Block protects the Not I site against nuclease activity. The TACA of the marker sequence helps todifferentiate the primer from naturally occurring Not I sites. The 8 random nucleotides are next. The trailing T is an artifactof the old chemistry when a specific nucleotide was used on the synthesis column. Newer chemistry allows any nucleotide in that position.
It is important to gel purify this oligo, for a truncated form containing the random sequence but not the Not I site would impair the cloning of the cDNA fragments.