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Revision as of 03:28, 28 October 2010 (view source) Mulan 12 (Talk | contribs) (New page: == Module Ⅱ, group 4 == After getting and then diluting the primer, do Two-step PCR. PCR with Pyrobest DNA polymerase. PCR system(50ul): 10×Pyrobest buffer Ⅱ 5ul ...) ← Older edit		Revision as of 03:54, 28 October 2010 (view source) Mulan 12 (Talk | contribs) (→Module Ⅱ, group 4) Newer edit →
Line 16:		Line 16:
	Total                             50μl		Total                             50μl
-	P+K+C:	+	PCR program:
-	H2O         0-10.5μl	+	Step                  Condition              Time
-	buffer BamHI 2μl	+	1                        95℃                5min
-	P+K+C         5-16μl	+	2                        95℃                30s
-	KpnI         1.5μl	+	3                        58℃                30s
-	BamHI         1μl	+	4                        72℃                1min
-	Total         20μl	+	5                 return to step2            15cycles
		+	6                        72℃                5min
		+	7                  add amplification primers
		+	8                        95℃                5min
		+	9                        95℃                30s
		+	10                        56℃                30s
		+	11                        72℃                1min
		+	12                return to step2            30cycles
		+	13                        72℃                1min
		+	14                        4℃                HOLD
-	Run a gel. Order: P+E+M 1-6 double digestion, Marker, P+K+C 1-6 double digestion, P+K+C PCR.	+
		+	Run a gel.
	== result ==		== result ==

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Revision as of 03:54, 28 October 2010

Module Ⅱ, group 4

After getting and then diluting the primer, do Two-step PCR.

PCR with Pyrobest DNA polymerase.

PCR system(50ul):

 10×Pyrobest buffer Ⅱ              5ul  
 Pyrobest	                    0.3ul  
 dNTPmix                            1ul
 Primer1                            1μl
 Primer2                            1ul
 Template DNA                      0.5ul
 Mgcl2                             0.5ul
 ddH2O                            40.5ul
 Total	                             50μl

PCR program:

 Step                  Condition              Time	        
 1                         95℃                5min
 2                         95℃                30s
 3                         58℃                30s
 4                         72℃                1min
 5                  return to step2            15cycles  
 6                         72℃                5min
 7                  add amplification primers  
 8                         95℃                5min
 9                         95℃                30s 
 10                        56℃                30s
 11                        72℃                1min
 12                 return to step2            30cycles
 13                        72℃                1min
 14                         4℃                HOLD

Run a gel.

result

P+E+M② is positive. It seems that PKC 1-6 are positive. But it still remains unclear whether the PCR bands are so weak and why the digestion result seemed negative.