Matt:LabNotes/2013-6-26: Difference between revisions
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====First Strand cDNA synthesis==== | |||
* Followed NEB E6300S protocol: [https://www.neb.com/protocols/1/01/01/first-strand-cdna-synthesis-e6300] | |||
Got 2ul aliquot of Human Brain Reference RNA from Blue at concentration 1ug/ul | |||
* Mixed components in two 0.2 ml microfuge tubes labelled HBBR 1ug cDNA | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
| align="center" style="background:#f0f0f0;"|'''2X Volume''' | |||
|- | |||
| RNA||1 ul (1 ug)||2 ul (2 ug) | |||
|- | |||
| d(T)23VN (50uM)||2 ul||4 ul | |||
|- | |||
| H2O||5 ul||10 ul | |||
|- | |||
| Total||8 ul||16 ul | |||
|- | |||
| | |||
|} | |||
* Denatured RNA for 5 min at 70C and then put on ice | |||
** Tightened the thermocycler too much and bent the tube slightly out of shape | |||
* Added the following to each tube | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|''' Components ''' | |||
| align="center" style="background:#f0f0f0;"|''' Volume ''' | |||
|- | |||
| M-MuLV Reaction Mix||10 ul | |||
|- | |||
| M-MuLV Enzyme Mix||2 ul | |||
|- | |||
| Total||20 ul | |||
|} | |||
* Incubated for one hour at 42C | |||
* Inactivated enzyme for 5 min at 80C | |||
* Kept on ice | |||
====Second Strand cDNA synthesis==== | |||
*Follow NEB E6111S protocol: [https://www.neb.com/protocols/1/01/01/second-strand-cdna-synthesis-e6111] | |||
*Added 48 ul H20 to each tube | |||
*Added 8ul 10x Second strand synthesis reaction buffer | |||
*Added 4ul Second strand synthesis enzyme mix | |||
*Mixed by pipetting | |||
*Incubated for 2.5 hours at 16C | |||
*Purified using Qiagen minelute column (eluted 23ul into 1.5ml tube labelled HBRR cDNA) | |||
*Measured DNA conc with Nandrop: | |||
**___ul (__ug) sample left in each tube |
Revision as of 19:13, 26 June 2013
Page Size Selection of 0 gap probes
USER/DpnII digested 0 gap probes:
Noi made 11.36ug/~400ul [1] I made 10.58ug/180ul [2]
To run ~3ug/gel, I use ~100ul of probes that Noi made for each of the 4 gels
- Ran TBU gel at 220V for 30 min with samples and ladder prepared below
- Heated TBE buffer in microwave
- Denatured samples/ladder for 10 min at 75C before putting on cold rack for 1 min
- Size selected for brightest band below 200bp
Components | 1X Volume | 4X Volume |
Enzyme digested probes (~11.36/400ul) | 106 | 424 |
TBE-Urea Buffer 2X | 106 | 424 |
H2O | 0 | 0 |
Total | 212 | 848 |
Components | 1X Volume | 4X Volume |
10-bp DNA ladder (1 ug/ul) | 1 | 4 |
TBE-Urea Buffer 2X | 5 | 20 |
H2O | 4 | 16 |
total | 10 | 40 |
First Strand cDNA synthesis
- Followed NEB E6300S protocol: [3]
Got 2ul aliquot of Human Brain Reference RNA from Blue at concentration 1ug/ul
- Mixed components in two 0.2 ml microfuge tubes labelled HBBR 1ug cDNA
Components | Volume | 2X Volume |
RNA | 1 ul (1 ug) | 2 ul (2 ug) |
d(T)23VN (50uM) | 2 ul | 4 ul |
H2O | 5 ul | 10 ul |
Total | 8 ul | 16 ul |
- Denatured RNA for 5 min at 70C and then put on ice
- Tightened the thermocycler too much and bent the tube slightly out of shape
- Added the following to each tube
Components | Volume |
M-MuLV Reaction Mix | 10 ul |
M-MuLV Enzyme Mix | 2 ul |
Total | 20 ul |
- Incubated for one hour at 42C
- Inactivated enzyme for 5 min at 80C
- Kept on ice
Second Strand cDNA synthesis
- Follow NEB E6111S protocol: [4]
- Added 48 ul H20 to each tube
- Added 8ul 10x Second strand synthesis reaction buffer
- Added 4ul Second strand synthesis enzyme mix
- Mixed by pipetting
- Incubated for 2.5 hours at 16C
- Purified using Qiagen minelute column (eluted 23ul into 1.5ml tube labelled HBRR cDNA)
- Measured DNA conc with Nandrop:
- ___ul (__ug) sample left in each tube