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