IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-19: Difference between revisions
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*PCR system contains: | *PCR system contains: | ||
<pre> | <pre> | ||
1 µL | 1 µL p1 | ||
1 µL | 1 µL p2 | ||
1 µL | 1 µL p3 | ||
4 µL dNTP | 4 µL dNTP | ||
0.5 µL Taq | 0.5 µL Taq | ||
Line 149: | Line 149: | ||
End | End | ||
</pre> | </pre> | ||
==The above PCR Product Purification== | ==The above PCR Product Purification== | ||
*Use Transgen EasyPure PCR Purification Kit. | *Use Transgen EasyPure PCR Purification Kit. |
Latest revision as of 02:07, 31 August 2007
Tandem Ori-T by Qu Mingzhi, Ren Ze
mini-prep R-OriT, S-Orit (pSB1A2)
- using Transgen mini plasmid puriflication kit.
- 50µL after purflication
mini-prep double digesting test
- R-OriT, S-OriT digestion system contains(Test):
1 µl 10*H buffer 0.25 µl EcoRI 0.25 µl PstI 5 µl Plasmid 3.5 µl dH20 -------------------------- 10 µl Total
electrophoresis result
- From left to right:
- 1 Marker(DL2000 plus)
- 2-4 R-OriT / pSB1A2 @ EcoRI/PstI
- 5-7 S-OriT / pSB1A2 @ EcoRI/PstI
double digesting for R-OriT (Standard Assembly vector)
- R-OriT digestion system contains(vector):
4 µl 10*M buffer 1 µl EcoRI 1 µl XbaI 20 µl Plasmid 14 µl dH20 -------------------------- 40 µl Total
electrophoresis result before gel extraction
- from left to right:
- 1-2 R-OriT_pSB1A2 @ EcoRI/PstI
- 3 Marker(DL2000 Plus)
electrophoresis result after gel extraction
- from left to right:
- 3 R-OriT_pSB1A2 @ EcoRI/PstI
- 4 Marker(DL2000 Plus)
colony PCR Test for R751, pSC101(III)
- Test Lb- R751 plate, Lb- pSC101 have the correct plasmid.
- Test plate:LB- R751, Lb- pSB101, Tc+ R751-pSC101, Tc+ Dh5α-pSC101, Tc+ R751-pSC101, Amp+ Dh5α-R0040
- primer :R751 OriT primer, pSC101 primer.
- according to <Colony PCR STANDARD PROTOCOL>
- PCR system contains(each well):
0.5 µl Primer 1(100uM) 0.5 µl Primer 2 2 µl dNTP(2.5uM) 2.5 µl 10X Taq Buffer 0.25 µl Taq 19 µl dH20 1 µl template -------------------------- ~25 µl Total
- PCR program condition 1: 94℃ 5min, 94℃ 30s, 53℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
- PCR program condition 2: 94℃ 5min, 94℃ 30s, 57℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .
electrophorsis result
- top:from left to right
- 1-4 pSC101
- 5-8 R751
- 9-16 R751-pSC101
- 17 Marker(DL2000 plus)
- botton:from left to right
- 18-21 Dh5α-pSC101
- 22-23 Dh5α-R0040
Lock & Key by Yu Tao
Transformation Result: R0010<-J01008 and R0040.J01010->E0040.B0015
- All plates grow clones.
- Select 6 probable positive colonies from the R0010<-J01008 experimental plate, culture them in liquid LB overnight for mini-prep.
- Select 3 probable positive colonies from the R0040.J01010->E0040.B0015 experimental plate, culture them in liquid LB overnight for mini-prep.
- I am not so optimistic.
Mini-prep: R0010, R0040.J01010->E0040.B0015(including its negative control group) and R0010<-J01008(1-6)
- Using Transgen mini plasmid purification kit.
- 50uL per tube after purification, 1 tube per type of plasmids except 3 tubes for the R0010.
Mini-prep Double Digesting Test Result
- Digesting all plasmids above with EcoRI/PstI.
- Each digestion system contains:
1 µl 10*H buffer 0.25 µl EcoRI 0.25 µl PstI 3 µl Plasmid 5.5 µl ddH20 -------------------------- 10 µl Total
- 37℃ culutre for 3 hours.
- from left to right:
- R0010<-J01008-1 @ EcoRI/PstI
- R0010<-J01008-2 @ EcoRI/PstI
- R0010<-J01008-3 @ EcoRI/PstI
- R0010<-J01008-4 @ EcoRI/PstI
- R0010<-J01008-5 @ EcoRI/PstI
- R0010<-J01008-6 @ EcoRI/PstI
- R0010<-J01008-previously selected @ EcoRI/PstI
- marker (DL2000 Plus)
- R0010-1 @ EcoRI/PstI
- R0010-2 @ EcoRI/PstI
- R0010-3 @ EcoRI/PstI
- R0040.J01010->E0040.B0015 @ EcoRI/PstI
- R0040.J01010->E0040.B0015-negative control group @ EcoRI/PstI
- E0040.B0015 @ EcoRI/PstI
- Conclusion:
- No positive result from these 6 R0010<-J01008 clones.
- R0010 seems correct.
- There is something wrong with the R0040.J01010->E0040.B0015, the miniprep fails. In fact, during the miniprep the color of the precipitation is much yellower than the normal one.
PCR: J01008
- J01008-p1,p2.,p3 primer: stored as 100uM.
- PCR system contains:
1 µL p1 1 µL p2 1 µL p3 4 µL dNTP 0.5 µL Taq 5µL 10 X buffer 37.5µL dH20 -------------------------- 50 µl Total
- Primer final concentration 2uM.
- I prepare 2 systems for the trial of 2 anneal temperature.
- Add a drop of liquid paraffin to each system.
- PCR program setting:
Step1 94℃ 5min Step2 94℃ 30s Step3 55℃/53℃ 30s Step4 70℃ 30s Step5 Go to step 2 for 4 more times Step6 72℃ 10min End
The above PCR Product Purification
- Use Transgen EasyPure PCR Purification Kit.
- 50uL per tube after purification, 1 tube per product.
Electrophorsis Result
- from left to right:
- J01010 PCR product
- J01008-1 (previous) purified PCR product
- J01008-2-1 (55℃) PCR product
- J01008-2-2 (53℃) PCR product
- J01008-2-1 (55℃) purified PCR product
- J01008-2-1 (55℃) purified PCR product
- Conclusion: only the J01008-2-2 seems correct.
Double Digestion: J01008-2-1,2 and R0010
- Digesting J01008-2-1,2 with XbaI/PstI and R0010 with SpeI/PstI.
- Each digestion system contains:
For J01008-2-1,2: / For R0010: 4 µl 10*M / 4 µl 10*H 1 µl XbaI / 1 µl SpeI 1 µl PstI / 1 µl PstI 10 µl Plasmid / 20 µl Plasmid 20 µl ddH20 / 14 µl ddH20 4 µl BSA / 0 µl BSA ---------------------------------------------------- 40 µl Total
- 37℃ overnight.